GMP-Compliant Manufacturing of [68Ga]Ga-ATH001 for a Human Microdose Trial Targeting Platelet-Derived Growth Factor Receptor Beta (PDGFRβ)

GMP-Compliant Manufacturing of [68Ga]Ga-ATH001 for a Human Microdose Trial Targeting Platelet-Derived Growth Factor Receptor Beta (PDGFRβ) | 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 GMP-Compliant Manufacturing of [ 68 Ga]Ga-ATH001 for a Human Microdose Trial Targeting Platelet-Derived Growth Factor Receptor Beta (PDGFRβ) Melodie Ferrat, Antonios Tzorzakakis, Mohammad Mahdi Moein, Tetyana Tegnebratt, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8384416/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: The use of suitable radiotracers in positron emission tomography (PET) enables non-invasive quantification of tissue biomarkers in patients. Platelet-derived growth factor receptor beta (PDGFRβ) is a key mediator of fibrogenesis and tumour stroma biology, expressed on activated pericytes, fibroblasts, and cancer-associated stromal cells. These features make PDGFRβ an attractive target for imaging pathological tissue remodelling across fibrotic and malignant diseases. Results: Here, we report the development and clinical translation of [ 68 Ga]Ga-DOTA-Cys-ATH001 ([ 68 Ga]Ga-ATH001), a novel PET tracer targeting PDGFRβ. [ 68 Ga]Ga-ATH001 was successfully produced using an automated in-house sequence on the ModularLab PharmTracer synthesis module. The process achieved high radiochemical purity (97 ± 0.4%), specific activity (2.4 ± 0.1 GBq/mg at the time of administration), reproducibility, and batch-to-batch consistency, ensuring suitability for clinical application. Translation into routine GMP production enabled a clinical trial in which [ 68 Ga]Ga-ATH001 was administered to participants with liver metastases. Conclusion: A robust, cGMP-compliant automated synthesis of [ 68 Ga]Ga-ATH001 has been established, supporting its clinical translation as a novel PDGFRβ-targeting PET tracer for quantitative imaging of fibrogenesis. The ongoing IMAGINE-1 trial will evaluate PDGFRβ-PET for visualization of liver tumour stroma. Together, these applications position [ 68 Ga]Ga-ATH001 as a versatile tracer for assessing PDGFRβ-mediated pathophysiology across both fibrotic and oncologic indications. EU Trial registration: ClinicalTrials.gov, NCT, NCT06562361, Registered 14 August 2024, Study Details | NCT06562361 | A Microdose Trial Investigating Binding of [68Ga]Ga-DOTA-CYS-ATH001 in Healthy Subjects and Different Patient Groups. | ClinicalTrials.gov Clinical Trials Information system: CTIS, EU CT 2025-522290-11-00, Registered 29 September 2025, IMAGINE 1 | 2025-522290-11-00 | CTIS.eu Positron Emission Tomography (PET) [68Ga]Ga-ATH001 Radiopharmaceuticals Platelet-Derived Growth Factor Receptor Beta (PDGFRβ) Fibrogenesis Tumour stroma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Platelet-derived growth factor receptor β (PDGFRβ) is a transmembrane receptor tyrosine kinase that plays an important role in regulating various tissue functions, such as cell proliferation and extracellular matrix remodelling. PDGFRβ is activated upon binding of platelet-derived growth factor (PDGF) ligands, particularly PDGF-BB and PDGF-DD, which initiate intracellular signalling cascades that support mitogenic and anti-apoptotic responses as well as cytoskeletal reorganization.(1),(2) Dysregulated PDGFRβ signalling has been implicated in pathological processes such as fibrosis, atherosclerosis, and cancer.(3),(4) In the liver, PDGFRβ is crucial for fibrogenesis through the activation of hepatic stellate cells and the deposition of extracellular matrix, leading to fibrosis and cirrhosis.(5) In liver tumours, such as metastases, PDGFRβ is expressed by stromal cells in the tumour microenvironment (6), and PDGFRβ inhibition synergized with chemotherapy in mouse models of colorectal cancer liver metastases (7). Given its important role in both fibrosis and tumours, PDGFRβ has emerged as an attractive therapeutic and diagnostic target. PDGFRβ-directed monoclonal antibodies (8) have been developed and tested preclinically for antitumor activity,(9) while several tyrosine kinase inhibitors (TKIs) (10) with activity against PDGFRα/β are already approved or under clinical investigation.(11) Multi-target TKIs such as imatinib(12) and sunitinib(13) are approved in oncology, whereas nintedanib, a potent inhibitor of PDGFRα/β, VEGFR, and FGFR has been approved for idiopathic pulmonary fibrosis and other progressive fibrotic lung diseases.(14) These examples illustrate how PDGFRβ-targeted strategies are being pursued in both malignant and non-malignant indications, highlighting its potential as an imaging biomarker and therapeutic target.(15) However, patient stratification for PDGFRβ-targeted therapies requires reliable detection of receptor expression. Conventional biopsy-based methods, such as immunohistochemistry, remain the clinical standard; however, they are invasive, limited by sample availability, and susceptible to false negatives due to inter- and intra-tumoral heterogeneity.(16) One promising strategy for non-invasive assessment of PDGFRβ expression is molecular imaging, which can overcome the limitations of conventional biopsy-based methods by allowing dynamic, whole-body visualization of receptor expression and treatment response.(17) Among the available strategies developed for molecular imaging, Affibody molecules represent a new class of small, engineered scaffold proteins (6.5 kDa, three-helix bundle) with high thermal stability, rapid biodistribution, and excellent tissue penetration, making them well-suited for imaging applications compared to the larger antibodies.(18, 19) When used as PET tracers, Affibody molecules may significantly improve patient stratification and therapeutic monitoring, ultimately advancing precision medicine approaches targeting PDGFRβ. Previous work has demonstrated the potential of PDGFRβ-targeted Affibody molecules: Z09591, which binds human and murine PDGFRβ with sub-nanomolar affinity, enabled high-contrast visualization of PDGFRβ-expressing xenografts when labelled with 111 In (20), 18 F (21, 22) and 68 Ga (23). Based on these promising results, Z09591 was translated into a GMP-compliant format and re-designated as ATH001 for clinical development. The lead clinical candidate, [ 68 Ga]Ga-DOTA-Cys-ATH001 ([ 68 Ga]Ga-ATH001), is labelled with gallium-68 for PET, enabling high-resolution whole-body imaging. Given the role of PDGFRβ in fibrogenesis, we hypothesized that [ 68 Ga]Ga-ATH001 could serve as a sensitive PET tracer for the detection and monitoring of liver fibrosis. This hypothesis is currently being investigated in an ongoing clinical trial (EU CT 2023-506555-15). In addition, since PDGFRβ is a recognized biomarker of tumour stroma, the tracer is also being evaluated in a separate ongoing study involving patients with primary and secondary liver tumours (EU CT 2025-522290-11-00). To ensure consistent and reliable production of high-quality tracers for molecular imaging in both trials, we developed a GMP-compliant manufacturing process for [ 68 Ga]Ga-ATH001 using the ModularLab Pharmtracer synthesizer (Eckert & Ziegler). (24) Materials and Methods General [ 68 Ga]Ga-ATH001 was produced in a Class C environment using the GMP-compliant Modular-Lab PharmTracer synthesizer (Ecker & Ziegler). The synthesis module is located in a BBC-type hotcell (Comecer), with a product transfer line connecting it to a lead-shielded product vial hatch, allowing the product to be removed safely while minimizing operator radiation exposure. Both the hotcell and the product vial hatch maintained the same cleanroom classification as the surrounding laboratory environment. The preparation of the product vial was performed within a Class A laminar airflow microbiological safety cabinet (Ninolab), with a sterile product filter and a ventilation filter. The product vial is then transferred to the lead-shielded vial hatch, where the transfer line is attached to the inlet of the sterile product filter. Materials and equipment used for the synthesis of [ 68 Ga]Ga-ATH001 The DOTA-ATH001 peptide precursor (400 μg/vial), an Affibody molecule comprising a 59 amino acid sequence, was generated by Solid Phase Peptide Synthesis (in metal-free conditions) under full GMP compliance by Almac Sciences (Edinburgh, UK) for clinical radiolabelling. [ 68 Ga]Ga-ATH001 was produced using a GMP-compliant Modular-Lab PharmTracer system (Eckert & Ziegler) with the corresponding reagent kit (EZ-102) and single-use hardware cassette (C4-GA-PEP), both specifically designed for the production of 68 Ga-labeled peptides on the Modular-Lab PharmTracer platform. The synthesizer module was operated according to the manufacturer's operation manual (Eckert & Ziegler, Berlin, Germany). The cassette and reagent kit were prepared in accordance with the manufacturer's instructions. The Modular-Lab PharmTracer synthesis sequence was developed in-house at Karolinska Radiopharmacy, Karolinska University Hospital, based on the acetone-free 68 Ga-DOTA peptide sequence provided by Eckert & Ziegler. The 68 GaCl 3 eluate was obtained from an Eckert & Ziegler 1.85 GBq 68 Ge/ 68 Ga-generator (GalliaPharm, Eckert & Ziegler, Germany). Radiolabelling of [ 68 Ga]Ga-ATH001 The radiopharmaceutical [ 68 Ga]Ga-ATH001 was synthesized using gallium-68 ( 68 Ga t 1/2 : 68 min) obtained as 68 GaCl 3 eluate from a 68 Ge/ 68 Ga-generator (Eckert & Ziegler). The generator was eluted by passing 5 mL of 0.1 N HCl into a sterile 15 mL glass vial (Huayi). The eluate activity was measured using a dose calibrator (Capintec) to determine the initial starting activity. The eluate was then transferred to the fully automated Modular-Lab PharmTracer synthesis module (Eckert & Ziegler) via syringe-driven transfer under control of the Modular-Lab software and connected to a single-use disposable cassette. Prior to radiolabelling, 68 GaCl 3 was concentrated on a cation exchange resin. Radiolabelling was performed by chelating gallium-68 with the DOTA moiety of ATH001 (200 μL, 1 mg/mL), previously mixed with EtOH/ TraceSELECT H 2 O 1:1 (0.4 mL), buffer solution (0.4 mL; pH = 4) and TraceSELECT H 2 O (1 mL) at 65 °C for 10 minutes, yielding a specific activity (A s ) ≥ 160 MBq/mg. The crude [ 68 Ga]Ga-ATH001 reaction mixture was subsequently purified by solid-phase extraction (HLB light SPE cartridge Oasis, Waters), eluted with 50% ethanol, and formulated in sterile saline (0.9%). The final product solution was sterilized by filtration through a 0.22 μm sterile filter (Millex-GV 0.22 µm, Merck Millipore). Equipment used for the quality control of [ 68 Ga]Ga-ATH001 High-performance liquid chromatography (HPLC) was performed using an Agilent 1260 Infinity system (Agilent Technologies) equipped with a G7111B pump, G7129A vial sampler, and a G7130A integrated column compartment. The system was coupled to a VWR detector (G7114A) and a radio-detector Flow-RAM with a 1″ NaI PMT (LabLogic) and connected using blue tubing (0.25 mm, five wraps). Data acquisition and evaluation were carried out with Laura software (LabLogic). The analytical column used was a Poroshell 120 EC-C18 column (3 × 150 mm, 2.7 μm) protected by a Poroshell 120 EC-C18 fast guard column (3 × 5 mm, 2.7 μm), with a column volume of 1 mL. The mobile phases consisted of (A) 0.1% trifluoroacetic acid (TFA) in water and (B) 60% acetonitrile (ACN) in 0.1% TFA. The applied gradient method (Ga-ATH001-HPLC) started with 60% A and 40% B, which was held for 1.5 min, followed by a linear increase to 100% B over 9 min, maintained for 2 min, and returned to the initial conditions for re-equilibration, giving a total run time of 15 min. The flow rate was set at 0.4 mL/min, the column temperature was maintained at room temperature, and the injection volume was 50 μL. Seal wash was performed with 20% ethanol in Milli-Q water. UV detection was carried out at 220 nm. The pH of [ 68 Ga]Ga-ATH001 was determined using pH indicator strips 2.0-9.0 (VWR) or pH-meter (type 913, version 2.913.0210, Metrohm). The integrity of the filter was assessed using a bubble point test with custom-made equipment (010105280602-A, DM Automation). Gas chromatography (GC) was carried out on an Agilent 6850 system equipped with a flame ionization detector (FID) and controlled by Laura 6 software (LabLogic). A Res-Solv column (30 m × 0.53 mm i.d., 1.0 μm film) with an autoinjector was used. The split ratio was set to 1:80, and the injection volume was 2 μL. Helium served as the carrier gas, while hydrogen and synthetic air were used for the detector. The inlet temperature was maintained at 250 °C with an inlet pressure of 2.76 PSI. The oven temperature program was as follows: initial isotherm at 35 °C for 4 min, ramp to 100 °C at 80 °C/min and hold for 30 s, ramp to 220 °C at 80 °C/min and hold for 30 s, followed by cooling to 35 °C. The specific activity was a critical parameter for this product, defined as the ratio of the radioactivity of [⁶⁸Ga]Ga-ATH001 to the total mass of DOTA-Cys-ATH001 (ATH001) as described in Equation 1. It was expressed in GBq/mg and determined both at the end of synthesis (EOS) and at EOS plus shelf-life (60–90 minutes post-synthesis), see Table 1. The radioactivity of [ 68 Ga]Ga-ATH001 is measured in a dose calibrator. The mass of DOTA-Cys-ATH001 is determined by liquid chromatography with UV detection and comparison of the area counts from the product sample with those from a known amount of ATH001 precursor (using a linearity function). PET imaging in human A phase one study is currently ongoing and includes a cohort of participants with liver metastases and intrahepatic cholangiocarcinoma (EU CT 2025-522290-11-00). No definitive or preliminary data are available yet, as recruitment has only just begun. However, a representative administered activity was estimated at 200 MBq ± 10% (approximately 2.5 MBq/kg body weight). The scanning session began with a dynamic scan of the liver for about 45 minutes after injection of [ 68 Ga]Ga-ATH001. This was followed by whole-body static PET acquisitions at 60 minutes and a static scan over the liver at 135 minutes post-injection. The radiation exposure from the administered activity was below 6 mSv. Non-contrast CT scans are used for attenuation correction and anatomical co-registration of PET images, resulting in a total radiation exposure (PET and CT components) of around 13.5 mSv. Results The radiolabelling of [ 68 Ga]Ga-ATH001, illustrated in Fig. 1, was performed under metal-free conditions using a GMP-compliant automated synthesizer. The in-house developed synthesis sequence for [ 68 Ga]Ga-ATH001 Solution for Injection involved three main steps: ( 1 ) Trapping and pre-concentration of [ 68 Ga]Ga 3+ ; ( 2 ) Chelation of [ 68 Ga]Ga 3+ with the DOTA moiety of ATH001 precursor; ( 3 ) Purification, formulation and sterile filtration of the final product. The synthesis was performed in the following sequences in Fig. 2: The 68 GaCl 3 eluate collected in a sterile vial from the generator was withdrawn by the module and loaded onto a strong cationic exchange (SCX) cartridge. [ 68 Ga]Ga 3+ was retained on the cartridge, while potential 68 Ge contaminants were eluted to the waste. The retained 68 Ga was subsequently released into the reaction vessel of the cassette by eluting with 0.7 mL of 5M NaCl/0.13N HCl (pH < 2). The radiolabelling step was performed by complexation of [ 68 Ga]Ga 3+ with the DOTA chelator of the precursor DOTA-Cys-ATH001 (V = 200 µL, C = 1 mg/mL), in the reaction vessel containing a EtOH/H 2 O 1:1 mixture (V = 0.4 mL), buffer solution (V = 0.4 mL of sodium acetate pH = 4) prepared from the EZ-102 reagent kit according to the Eckert & Ziegler “user manual for synthesis of [ 68 Ga]-conjugated peptides with PharmTracer fractionation and pre-purifications by cation exchange” and TraceSELECT H 2 O (V = 1 mL). The reaction mixture had a pH of 3.5-4 and was heated at 65°C for 10 min (Fig. 1). The crude [ 68 Ga]Ga-ATH001 reaction mixture was diluted with sterile saline (9 mg/mL, 5 mL) prior to purification using a reversed phase solid phase extraction (SPE) cartridge (Oasis light HLB SPE within the EZ cassette). The product was eluted with an EtOH/H 2 O 1:1 mixture and formulated with sterile sodium chloride (9 mg/mL, reagent kit EZ-102). The final product was sterilized through a 0.22 µm sterile membrane filter and collected in a 15 mL sterile vial (Huayi) as [ 68 Ga]Ga-ATH001 Solution for Injection. Figure 1. Radiolabeling of [ 68 Ga]Ga-ATH001 Solution for Injection Figure 2. Flowchart of Modular-Lab (E&Z) module used for the synthesis of [ 68 Ga]Ga-ATH001 An automated GMP-compliant labelling process for [ 68 Ga]Ga-ATH001 was successfully implemented in which, three consecutive batches were produced under normal conditions and one batch was performed to control potential microbiological burden originated from the radio-synthesizer, yielding an average decay-corrected radiochemical yield of 49.8 ± 3.6% (n = 4), with an average final product activity of 429.3 ± 27.4 MBq (n = 4) and a radiochemical purity of 96.8 ± 0.4% among all batches. The mean specific activity at the estimated administration time (shelf-life included) was 2.43 ± 0.1 GBq/mg (n = 4). Stability studies have been performed where the radiochemical stability of [ 68 Ga]Ga-ATH001 was 96.8 ± 0.4% after 2 hours at room temperature (Table 1 ). Table 1 Process validation of [68Ga]Ga-ATH001 Parameters Product specification Batch 1 Batch 2 Batch 3 Batch 4 a Activity product at EOS in MBq (after withdrawal of QC and sterility samples) ≥ 160 MBq 452 MBq 431 MBq 450 MBq 384 MBq Volume of product in mL (after withdrawal of QC and sterility samples) ≤ 10 mL 7.28 mL 7.24 mL 7.14 mL 7.65 b mL Activity concentration (MBq/mL) - 62.05 MBq/mL 59.49 MBq/mL 63.06 MBq/mL 50.19 MBq/mL Appearance Clear, colourless or slightly yellow. Free of visual particles Comply Comply Comply Comply pH 4.0–8.0 5.0 5.0 5.0 4.5 Radionuclidic identity half-life 68 Ga 62 to 74 min 66 min 69 min 68 min 70 min Concentration of ATH001 (principal peak on SST UV) 1–20 µg/mL 9.97 µg/mL 8.87µg/mL 10.3 µg/mL 9.91 µg/mL Chemical impurity(ies) (peaks except principal peak detected on UV) 0–5% 3% 0% 0% 2% Product identification [Rt RD – Rt UV | ≤ 60 s 6 s 8 s 8 s 11 s Radiochemical purity HPLC ≥ 91% 97% 97% 97% 96% Specific activity EOS ≥ 1.6 GBq/mg 6.44 GBq/mg 6.76 GBq/mg 6.88 GBq/mg 5.91 GBq/mg Specific activity (EOS + shelf-life) ≥ 1.6 GBq/mg 2.28 GBq/mg 2.51 GBq/mg 2.45 GBq/mg 2.46 GBq/mg Filter integrity ≥ 3.5 bar 4.2 bar 4.1 bar 4.2 bar N.A a Sterility Sterile Sterile Sterile Sterile Sterile Bacterial endotoxins < 17.5 IU/mL < 5.00 IU/mL < 5.00 IU/mL < 5.00 IU/mL < 5.00 IU/mL Ethanol content ≤ 80 mg/mL 50 mg/mL 50 mg/mL 44 mg/mL 47 mg/mL Radiochemical Stability RCP ≥ 91% after 120 minutes 96% 97% 96% 96% Abbreviations: Rt RD = retention time from radio-detector; Rt UV = retention time from UV detector, EOS = end of synthesis. a Microbiological worst-case challenge: a batch in which the sterile filter is intentionally excluded. b A larger product volume is observed due to the omission of the sterile filter. Quality control Validation of the analytical methods confirmed that the procedures are reliable and appropriate for their intended purpose. The results of quality control testing of all product validation batches are summarized in Table 1 . Representative chromatograms from HPLC, and GC analyses are presented in Figs. 3, and 4, respectively. The HPLC chromatogram of [⁶⁸Ga]Ga-ATH001 (Fig. 3) presents a dominant peak at Rt ≈ 8.53 min as well as the UV peak of the non-radioactive precursor (UV Rt ≈ 8.45 min). [ 68 Ga]Ga-ATH001 has an RCP of ~ 96.8%, with traces of 68 Ga-ions (RCP ≤ 1.3% within the region of interest (ROI). The total RCP of 68 Ga-related impurities was ≤ 1.8%, which is consistent with the specifications (Table 1 ). The UV peak corresponding to ATH001 had an integrated area with a concentration of 11.25 µg·mL⁻¹, as determined from the previously established calibration curve. This concentration was used to calculate the injected molar amount and the specific activity, which is a critical parameter for the planned microdose study. Figure 3. Representative HPLC chromatograms of [ 68 Ga]Ga-ATH001 at EOS. [ 68 Ga]Ga 3+ ions and other radiochemical impurities may be retained in the injection system, tubing or the column material itself. Several approaches exist to assess these retention effects and must be considered during method validation. To address this, we compared the injected radioactivity with that recovered in the eluent. This can be achieved by collecting the eluent in fractions and measuring their radioactivity, then comparing these values with the injected activity.( 25 ) To omit the additional iTLC procedure, which is time-consuming and may negatively impact the specific activity of the final product due to time delay for batch release, a column recovery has been performed where the radioactivity of the QC sample (200 µL) was first measured with a Capintec dose calibrator, applying appropriate decay time correction. Subsequently, 50 µL of the product was injected onto the HPLC column. During the 15-minute HPLC run, all eluate from the column was collected, and its radioactivity measured using the dose calibrator. The residual activity remaining in the HPLC injection vial was also recorded. By comparing the time-corrected total activity recovered from the column eluate plus the residual vial activity to the initial activity in the product vial, the recovery percentage was calculated and found to be in the range of 90–110%, confirming near-complete elution of the radiolabelled compound. The HPLC recovery is calculated following the Eq. 2 in-below: Equation 2 : \(\:Recovery\:\left(\%\right)=\frac{\text{A}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:\text{l}\text{e}\text{f}\text{t}\:\text{i}\text{n}\:\text{v}\text{i}\text{a}\text{l}\:\text{a}\text{f}\text{t}\text{e}\text{r}\:\text{i}\text{n}\text{j}\text{e}\text{c}\text{t}\text{i}\text{o}\text{n}+\text{A}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:\text{c}\text{o}\text{l}\text{l}\text{e}\text{c}\text{t}\text{e}\text{d}\:\text{f}\text{r}\text{o}\text{m}\:\text{c}\text{o}\text{l}\text{u}\text{m}\text{n}}{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}\:\text{i}\text{n}\:\text{v}\text{i}\text{a}\text{l}\:\text{b}\text{e}\text{f}\text{o}\text{r}\text{e}\:\text{i}\text{n}\text{j}\text{e}\text{c}\text{t}\text{i}\text{o}\text{n}}*100\) The GC analysis of the final product showed a single ethanol peak (Rt ~ 2.5min) and only trace/LLQ signal for acetone (Fig. 4). Quantification of the ethanol calibration curve gave an ethanol content that met our predefined release limit and is compliant with Class-3 solvent expectations (ICH Q3C). No additional volatile impurities were detected above the reporting threshold. Therefore, the ethanol level of the formulation is within acceptable limits for clinical use and aligns with its role as a stabilizer agent. Figure 4. Representative GC chromatograms of [ 68 Ga]Ga-ATH001 Discussion An automated GMP-compliant process for [ 68 Ga]Ga-ATH001 was successfully implemented on the ModularLab synthesis module, with high radiochemical purity 96.8 ± 0.4% (n = 4) and high specific activity (2.43 ± 0.1 GBq/mg at the time of administration), in compliance with microdosing requirements. Stability tests of [ 68 Ga]Ga-ATH001 demonstrated that the radiochemical purity remained ≥ 96% for up to 2 hours at room temperature with no trace of radiolysis, thus eliminating the need for additional stabilizers. These results are comparable to the automated process developed by Jussing et al. for a different Affibody molecule-based PET tracer [ 68 Ga]Ga-ABY-025,( 26 ) where validation batches achieved high yields, purity, and stability while using ModularLab synthesis module, including a worst-case batch without sterile filtration to study microbiological robustness. These findings demonstrate that the automated 68 Ga-labeling process is both reliable and applicable to translational and clinical use. Following the findings on [ 68 Ga]DOTA-Z09591 ( 23 ), our fully automated process provides harmonization, GMP reproducibility, and optimized precursor use achieving high specific activity without radiolysis. In contrast, 18 F-labeling strategies such as [ 18 F]TZ-Z09591( 21 ) require two-steps approach, involving radiosynthesis of a prosthetic tetrazine group followed by a TCO-tetrazine click chemistry ligation to the Affibody molecule. While efficient, this resulted in moderate molar activity (~ 20 MBq/nmol) and increased operational complexity. Thus, although 18 F provides advantages due to its longer half-life and the possibility of large-scale production, the use of 68 Ga is particularly attractive for clinical studies because of its on-site generator availability, well-established clinical use, and straightforward DOTA chemistry.( 27 ) Moreover, alternative chelation strategies, such as RESCA for Al- 18 F labelling, have shown inferior in vivo stability compared to DOTA or NOTA, with the evidence of Ga-RESCA trans-chelation under serum and acidic conditions. This may reduce imaging clarity and increase off-target radiation.( 21 , 23 ) This reduced robustness, combined with more limited clinical validation, makes 68 Ga-DOTA chemistry the more reliable choice for early clinical development.( 26 ) The selection of PDGFRβ as the biological target for an imaging agent offers various advantages over alternative strategies such as fibroblast activation protein (FAP), another emerging PET-compatible stromal marker. While FAP-targeted tracers are of strong interest for their high tumour-to-background ratios, particularly in desmoplastic tumours,( 28 , 29 ) PDGFRβ represents a more direct and clinically validated target. Several PDGFRβ inhibitors, including imatinib( 30 ), sunitinib( 31 ) and pazopanib, are FDA-approved and used in oncology. Moreover, PDGFRβ itself is frequently expressed on tumour cells and pericytes, as well as cancer-associated fibroblasts in the tumour microenvironment, potentially enabling direct visualization of tumour biology and angiogenesis rather than solely stromal components alone.( 19 , 23 ) Furthermore, PDGFR signalling has been extensively studied in oncogenesis, fibrosis, and angiogenesis, providing strong mechanistic support for imaging applications, with isoform-specific biology (PDGFRα vs PDGFRβ) allowing additional precision. Despite potential limitations such as physiological expression in normal tissues and the risk of resistance mechanisms, PDGFRβ remains a well-validated therapeutic target, potentially providing a more direct translational pathway than FAP for early diagnostic development. The optimized automated production of [ 68 Ga]Ga-ATH001 on a ModularLab synthesizer, with a RCY of ≥ 49.8% (n = 4) and a RCP of ≥ 96.8%, provide a high-quality tracer with excellent reproducibility in compliance with GMP specifications for a clinical trial. Further improvements could include the use of cyclotron-produced 68 Ga to achieve higher specific activity of [ 68 Ga]Ga-ATH001. Recent advances in solid-target cyclotron production with optimized purification methods (e.g., ascorbate-assisted UTEVA resin processing) have enabled apparent molar activities approaching 500 GBq/µmol and batch yields up to 7 GBq, representing a ten-fold increase compared to generator-based production.( 32 ) Such developments could facilitate large-scale multi-center trials and broaden clinical accessibility. This positions [ 68 Ga]Ga-ATH001 as a clinically translatable PET tracer that not only builds on prior experience with 68 Ga-labeled Affibody molecules, ( 23 ) but also overcomes limitations of 18 F-based approaches ( 21 ) and provides advantages over emerging alternatives such as FAPI. Clinical PET/CT scanning The [ 68 Ga]Ga-ATH001 PET/CT examination was performed on a GE Discovery DMI generation 2 (Milwaukee, WI, USA) scanner. Representative data from PET/CT images of [ 68 Ga]Ga-ATH001 in a study participant with a liver metastasis from colorectal cancer are shown in Fig. 5. The liver metastasis exhibited increased tracer uptake compared with the surrounding healthy liver tissue, even at early time points. Figure 5. [ 68 Ga]Ga-ATH001 PET/CT of a hepatic metastatic lesion Representative images from a study participant with a large metastatic lesion in liver segments 6/7 are shown on contrast-enhanced axial CT (Fig. 5a). [ 68 Ga]Ga-ATH001 PET/CT examination reveals increased tracer uptake (arrows) compared with the surrounding liver parenchyma at both early (Fig. 5b,c) and late (Fig. 5d,e) time points. Conclusions The GMP-compliant automated synthesis of [ 68 Ga]Ga-ATH001 consistently delivers a high-quality PET tracer suitable for clinical use. This advancement enables non-invasive characterization of PDGFRβ expression in tissue and supports progress in both cancer- and fibrosis-related research, as well as anti-fibrotic drug development. Further improvements could include the use of cyclotron-produced 68 Ga to increase batch activity, enhance production scalability, and allow imaging of multiple participants from a single synthesis of [ 68 Ga]Ga-ATH001. The ongoing IMAGINE-1 trial will evaluate PDGFRβ-PET for visualization of tumour stroma and angiogenesis in participants with liver tumours and intrahepatic cholangiocarcinoma, expanding the clinical applications of [ 68 Ga]Ga-ATH001 across oncologic and fibrotic indications. Abbreviations - ACN Acetonitrile - Al- 18 F Aluminium-[ 18 F]Fluoride - As Specific activity (GBq/mg) - cGMP Current Good Manufacturing Practices - CT Computed tomography - DOTA 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid - EOS End of Synthesis - EtOH Ethanol - E&Z Eckert & Ziegler - FAP Fibroblast activation protein - FGFR Fibroblast Growth Factor Receptor - FID Flame ionization detector - [ 68 Ga]Ga-ATH001 [ 68 Ga]Ga-DOTA-Cys-ATH001 - GC Gas chromatography - GMP Good manufacture practices - H 2 O Water - HCl Hydrochloric acid - HLB Hydrophilic-lipophilic balance cartridge - HPLC High-performance liquid chromatography - IMAGINE Imatinib Augmented Perfusion for GastroIntestinal Liver Neoplasia - iTLC instant Thin layer chromatography - NaCl Sodium chloride - NOTA 1,4,7-triazacyclononane-1,4,7-triacetic acid - PET Positron emission tomography - PDGFRβ Platelet-derived growth factor receptor beta - PSI Pound-force per square inch – Unit of pressure - QC Quality control - RCP Radiochemical purity (%) - RCP Tot Total radiochemical purity - RCY Radiochemical yield (%) - RD Radio-detector - RESCA Rapid, Efficient and Stable chelator for Aluminium-[ 18 F]Fluoride - ROI Region of interest - Rt Retention time - Rt RD Retention time from radio-detector - Rt UV Retention time from UV detector - SCX Strong cationic exchange cartridge - TFA Trifluoroacetic acid - TKI Tyrosine kinase inhibitors - UV Ultra-violet - VEGFR Vascular Endothelial Growth Factor Receptor Declarations Ethical approval and consent to participate The clinical PET imaging study was approved by the Swedish Ethical Review Authority under EU CT 2025-522290-11-00,and all participants gave written consent before initiating any study procedure. Consent for publication The clinical patient data used in the publication have been anonymized and safeguards measures taken to follow the GDPR (General Data Protection Regulation) requirements according to the Karolinska University Hospital’s regulation. Availability of data and material Summary data supporting the findings of the current study are included within the article. Raw PET imaging data are not publicly available due to ethical and participant privacy considerations but may be available from the corresponding author upon reasonable request and subject to appropriate approval. Funding The GMP validation was funded by Antaros Tracer AB. The precursor materials were supplied by Antaros Tracer AB, under license from Affibody AB. The First-in-Human microdose study (2023-506555-15) was funded by Takeda Pharmaceutical Company Limited and Antaros Tracer AB, whereas the clinical program (2025-522290-11-00) described in this paper is supported by funding from Radiumhemmet´s Research Fund, Karolinska Institutet’s Microenvironmental Control of Metastasis network, and a private donor. Competing interests Ayman Abouzayed, Per Hagmar and Olof Eriksson are employees at Antaros Tracer AB. Talakad Lohith is an employee at Takeda Pharmaceuticals Company Limited. Olof Eriksson is mentioned as inventor of a patent relating to PDGFRβ binding peptides. The remaining authors declare no conflicts of interest. Author’s contribution All authors contributed to the study conception and design. Mélodie Ferrat, Mohammad Mahdi Moein, Tetyana Tegnebratt, Emma Jussing and Thuy Tran were responsible for the GMP validation and manufacturing of [ 68 Ga]Ga-ATH001. Ayman Abouzayed, Per Hagmar and Olof Eriksson designed and developed the peptide precursor. Mélodie Ferrat developed the radiolabelling procedure in the GMP laboratory and was the major contributor in writing the manuscript. Rimma Axelsson, Antonios Tzorzakakis, Marco Gerling and Jennie Engstrand, contributed with expertise regarding the clinical examination for which the radiotracer production is intended. All authors contributed to reading and approving the manuscript. Acknowledgement We would like to thank all members part of this study for their help and contribution as well as Karolinska Radiopharmacy group for their support. References Friedman SL, Sheppard D, Duffield JS, Violette S. Therapy for Fibrotic Diseases: Nearing the Starting Line. Science translational medicine. 2013;5(167):167sr1. Dobie R, Connelly J, Henderson NC. PDGF-Mediated Regulation of Liver Fibrosis. Current pathobiology reports. 2015;3(4):225–33. Heldin C-H. Targeting the PDGF Signaling Pathway in the Treatment of Non-Malignant Diseases. Journal of neuroimmune pharmacology. 2014;9(2):69–79. Heldin C-H. Targeting the PDGF signaling pathway in tumor treatment. Cell communication and signaling. 2013;11(1):97–. Heldin CH, Westermark B. Platelet-derived growth factor: mechanism of action and possible in vivo function. Cell regulation. 1990;1(8):555–66. Fernández Moro C, Geyer N, Harrizi S, Hamidi Y, Söderqvist S, Kuznyecov D, et al. An idiosyncratic zonated stroma encapsulates desmoplastic liver metastases and originates from injured liver. Nat Commun. 2023;14(1):5024. Kitadai Y, Sasaki T, Kuwai T, Nakamura T, Bucana CD, Fidler IJ. Targeting the expression of platelet-derived growth factor receptor by reactive stroma inhibits growth and metastasis of human colon carcinoma. Am J Pathol. 2006;169(6):2054–65. Ogawa S, Ochi T, Shimada H, Inagaki K, Fujita I, Nii A, et al. Anti-PDGF-B monoclonal antibody reduces liver fibrosis development. Hepatology research. 2010;40(11):1128–41. Shen J, Vil MD, Prewett M, Damoci C, Zhang H, Li H, et al. Development of a fully human anti-PDGFRbeta antibody that suppresses growth of human tumor xenografts and enhances antitumor activity of an anti-VEGFR2 antibody. Neoplasia. 2009;11(6):594–604. Stacchiotti S, Longhi A, Ferraresi V, Grignani G, Comandone A, Stupp R, et al. Phase II Study of Imatinib in Advanced Chordoma. Journal of clinical oncology. 2012;30(9):914–20. Dai Y. Platelet-derived growth factor receptor tyrosine kinase inhibitors: a review of the recent patent literature. Expert Opin Ther Pat. 2010;20(7):885–97. FDA. Drug approval: Gleevec (Imatinib Mesylate) Capsules 2001 [Available from: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2001/21335_Gleevec.cfm#:~:text=Approval%20Date:%205/10/,Part%202%20(PDF). (FDA) USFaDA. Drug Approval: Sutent (Sunitinib Malate) Capsules 2006 [Available from: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2006/021938s000_ 021968s000_Stutent.cfm#:~:text=Approval%20Date:%2001/26/,s)%20&%20 Correspondence%20(PDF). Fala L. Ofev (Nintedanib): First Tyrosine Kinase Inhibitor Approved for the Treatment of Patients with Idiopathic Pulmonary Fibrosis. Am Health Drug Benefits. 2015;8(Spec Feature):101–4. Ying HZ, Chen Q, Zhang WY, Zhang HH, Ma Y, Zhang SZ, et al. PDGF signaling pathway in hepatic fibrosis pathogenesis and therapeutics (Review). Mol Med Rep. 2017;16(6):7879–89. Friedman SL, Pinzani M. Hepatic fibrosis 2022: Unmet needs and a blueprint for the future. Hepatology (Baltimore, Md). 2022;75(2):473–88. Tolmachev V, Stone-Elander S, Orlova A. Radiolabelled receptor-tyrosine-kinase targeting drugs for patient stratification and monitoring of therapy response: prospects and pitfalls. The lancet oncology. 2010;11(10):992–1000. Löfblom J, Feldwisch J, Tolmachev V, Carlsson J, Ståhl S, Frejd FY. Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS letters. 2010;584(12):2670–80. Lindborg M, Cortez E, Höidén-Guthenberg I, Gunneriusson E, von Hage E, Syud F, et al. Engineered High-Affinity Affibody Molecules Targeting Platelet-Derived Growth Factor Receptor β In Vivo. Journal of molecular biology. 2011;407(2):298–315. Tolmachev V, Varasteh Z, Honarvar H, Hosseinimehr SJ, Eriksson O, Jonasson P, et al. Imaging of platelet-derived growth factor receptor β expression in glioblastoma xenografts using affibody molecule 111In-DOTA-Z09591. The Journal of nuclear medicine (1978). 2014;55(2):294. Wegrzyniak O, Zhang B, Rokka J, Rosestedt M, Mitran B, Cheung P, et al. Imaging of fibrogenesis in the liver by [18F]TZ-Z09591, an Affibody molecule targeting platelet derived growth factor receptor β. EJNMMI radiopharmacy and chemistry. 2023;8(1):23–18. Wegrzyniak O, Lechi F, Mitran B, Cheung P, Bitzios A, Persson J, et al. Non-invasive PET imaging of liver fibrogenesis using a RESCA-conjugated Affibody molecule. iScience. 2024;27(5):109688. Strand J, Varasteh Z, Eriksson O, Abrahmsen L, Orlova A, Tolmachev V. Gallium-68-Labeled Affibody Molecule for PET Imaging of PDGFRβ Expression in Vivo. Molecular pharmaceutics. 2014;11(11):3957–64. Lubberink M, editor Dosimetry and safety of the PDGFRb PET tracer [68Ga]GaDOTA-Cys-ATH001: first-in-human data in patients with liver fibrosis and healthy subjects. Annual Congress of the European Association of Nuclear Medicine (EANM) 2025; 2025; Barcelona, Spain: Elsevier. Gillings N, Todde S, Behe M, Decristoforo C, Elsinga P, Ferrari V, et al. EANM guideline on the validation of analytical methods for radiopharmaceuticals. EJNMMI Radiopharmacy and Chemistry. 2020;5(1):7. Jussing E, Ferrat M, Moein MM, Alfredéen H, Tegnebratt T, Bratteby K, et al. Optimized, automated and cGMP-compliant synthesis of the HER2 targeting [68Ga]Ga-ABY-025 tracer. EJNMMI radiopharmacy and chemistry. 2023;8(1):41–18. Fani M, André JP, Maecke HR. 68Ga-PET: a powerful generator-based alternative to cyclotron-based PET radiopharmaceuticals. Contrast media and molecular imaging. 2008;3(2):53–63. Lindner T, Loktev A, Altmann A, Giesel F, Kratochwil C, Debus J, et al. Development of Quinoline-Based Theranostic Ligands for the Targeting of Fibroblast Activation Protein. Journal of Nuclear Medicine. 2018;59(9):1415–22. Kratochwil C, Flechsig P, Lindner T, Abderrahim L, Altmann A, Mier W, et al. 68 Ga-FAPI PET/CT: Tracer Uptake in 28 Different Kinds of Cancer. Journal of Nuclear Medicine. 2019;60(6):801–5. Demetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ, et al. Efficacy and Safety of Imatinib Mesylate in Advanced Gastrointestinal Stromal Tumors. The New England journal of medicine. 2002;347(7):472–80. Motzer RJ, Hutson TE, Tomczak P, Michaelson MD, Bukowski RM, Rixe O, et al. Sunitinib versus Interferon Alfa in Metastatic Renal-Cell Carcinoma. The New England journal of medicine. 2007;356(2):115–24. Jussing E, Milton S, Samén E, Moein MM, Bylund L, Axelsson R, et al. Clinically Applicable Cyclotron-Produced Gallium-68 Gives High-Yield Radiolabeling of DOTA-Based Tracers. Biomolecules. 2021;11(8):1118. 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. 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-8384416","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":563943454,"identity":"333e9c5f-c105-4321-a750-48d77cb8573f","order_by":0,"name":"Melodie Ferrat","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIie3PsWrCQBzH8X/4w93ywzmg2CconBxkCumrnAg3+wiKYBZ9gEKhrxIJmKW0q+BgsuiSwdGCoElb2u107HDf6Q7uw//+RD7fv4wnBEUkJ1+3WInbJPgmyNqLsfcS+iW5ugke0+m0rMcxQS73x8/Th+6QrEoXid5Ws8GLsgQU+nlhtpEgaOeoaDOcd6FyegotE8w2bogInWRXteRCeNhzcDbvDZGHk3tK0JKMEApmmKz5GEUu0ewybHcZAbDMPTvSgqHdHyvyVVmfkz7kmoM6TgavaVodnWN+wt+R73nv8/l8PmdXfyU+RvPx7EUAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7004-3565","institution":"Karolinska Institute: Karolinska Institutet","correspondingAuthor":true,"prefix":"","firstName":"Melodie","middleName":"","lastName":"Ferrat","suffix":""},{"id":563943455,"identity":"72d49276-125a-46ce-a979-857d8f581d4c","order_by":1,"name":"Antonios Tzorzakakis","email":"","orcid":"","institution":"Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Antonios","middleName":"","lastName":"Tzorzakakis","suffix":""},{"id":563943456,"identity":"5dd6e0ab-77e8-4689-912e-bd5d7dda9052","order_by":2,"name":"Mohammad Mahdi Moein","email":"","orcid":"","institution":"Karolinska University Hospital: Karolinska Universitetssjukhuset","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Mahdi","lastName":"Moein","suffix":""},{"id":563943457,"identity":"8cd2b54c-d92e-46f6-a939-60866cd4985c","order_by":3,"name":"Tetyana Tegnebratt","email":"","orcid":"","institution":"Karolinska University Hospital: Karolinska Universitetssjukhuset","correspondingAuthor":false,"prefix":"","firstName":"Tetyana","middleName":"","lastName":"Tegnebratt","suffix":""},{"id":563943458,"identity":"0dddb7f7-4a34-4f80-a176-9569941014b5","order_by":4,"name":"Ayman Abouzayed","email":"","orcid":"","institution":"Antaros Tracer AB","correspondingAuthor":false,"prefix":"","firstName":"Ayman","middleName":"","lastName":"Abouzayed","suffix":""},{"id":563943459,"identity":"12e37fc3-4e36-4b06-95e3-e931cd775f3b","order_by":5,"name":"Talakad Lohith","email":"","orcid":"","institution":"Takeda Pharmaceuticals America Inc","correspondingAuthor":false,"prefix":"","firstName":"Talakad","middleName":"","lastName":"Lohith","suffix":""},{"id":563943460,"identity":"9d7d2839-b693-4b6f-add8-0a4564cf7777","order_by":6,"name":"Per Hagmar","email":"","orcid":"","institution":"Antaros Tracer AB","correspondingAuthor":false,"prefix":"","firstName":"Per","middleName":"","lastName":"Hagmar","suffix":""},{"id":563943461,"identity":"dcef73d3-5668-4e5e-98b0-af9610a45c61","order_by":7,"name":"Emma Jussing","email":"","orcid":"","institution":"Karolinska Institute: Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Emma","middleName":"","lastName":"Jussing","suffix":""},{"id":563943462,"identity":"cd25fbf0-76b2-4e92-9139-32cdebbf235b","order_by":8,"name":"Rimma Axelsson","email":"","orcid":"","institution":"Karolinska Institute - Huddinge Campus: Karolinska Institutet - Huddinge Campus","correspondingAuthor":false,"prefix":"","firstName":"Rimma","middleName":"","lastName":"Axelsson","suffix":""},{"id":563943463,"identity":"a89f5e0a-2389-4657-9b1f-c23dac7be551","order_by":9,"name":"Jennie Engstrand","email":"","orcid":"","institution":"Karolinska Institute: Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Jennie","middleName":"","lastName":"Engstrand","suffix":""},{"id":563943464,"identity":"b1812a6f-044e-407f-b2b7-13cd0f9e64b4","order_by":10,"name":"Marco Gerling","email":"","orcid":"","institution":"Karolinska Institute: Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Gerling","suffix":""},{"id":563943465,"identity":"772b50b2-cd75-44f9-8147-3446e3245c94","order_by":11,"name":"Olof Eriksson","email":"","orcid":"","institution":"Antaros tracer AB","correspondingAuthor":false,"prefix":"","firstName":"Olof","middleName":"","lastName":"Eriksson","suffix":""},{"id":563943466,"identity":"abbd8451-f091-44be-b878-6d28b11c1ef6","order_by":12,"name":"Thuy A. Tran","email":"","orcid":"","institution":"Karolinska Institute: Karolinska Institutet","correspondingAuthor":false,"prefix":"","firstName":"Thuy","middleName":"A.","lastName":"Tran","suffix":""}],"badges":[],"createdAt":"2025-12-17 10:10:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8384416/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8384416/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99192228,"identity":"cc53ae30-7c95-4caf-b38a-2c935990c4cc","added_by":"auto","created_at":"2025-12-30 01:03:15","extension":"xml","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":15877,"visible":true,"origin":"","legend":"","description":"","filename":"erpcERPCD2500165.xml","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/3ad51c06a7d2905327aad384.xml"},{"id":99316799,"identity":"5b2ebadb-f5bd-4b88-b51b-5553bc60d4c1","added_by":"auto","created_at":"2025-12-31 16:29:13","extension":"xml","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1112,"visible":true,"origin":"","legend":"","description":"","filename":"ERPCD25001654175.go.xml","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/f21e11bab42b1c83a8753c4e.xml"},{"id":99316754,"identity":"fb9c41db-c9d6-46df-aa4e-dabb999788fe","added_by":"auto","created_at":"2025-12-31 16:29:07","extension":"xml","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":951,"visible":true,"origin":"","legend":"","description":"","filename":"ERPCD2500165Import.xml","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/9cbb0f7c57d3a2da287bba66.xml"},{"id":99192233,"identity":"00c72667-50c1-41bf-b385-7437f599dd90","added_by":"auto","created_at":"2025-12-30 01:03:16","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107757,"visible":true,"origin":"","legend":"","description":"","filename":"ERPCD25001650enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/d73deef0795f9eba222afe8d.xml"},{"id":99317545,"identity":"c5e4e01c-eb8c-4d9d-9644-d9e446c5f355","added_by":"auto","created_at":"2025-12-31 16:30:21","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":59782,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/5f22dbc634919c654ac0f393.pdf"},{"id":99192234,"identity":"9b32388e-bc1b-4123-8eec-14534628fa67","added_by":"auto","created_at":"2025-12-30 01:03:16","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":111588,"visible":true,"origin":"","legend":"","description":"","filename":"Figure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/094babc35a4ad49e321e6cd4.pdf"},{"id":99192236,"identity":"7455d132-3419-4c91-8354-4a24a19f0aa2","added_by":"auto","created_at":"2025-12-30 01:03:16","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95854,"visible":true,"origin":"","legend":"","description":"","filename":"Figure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/7aa6443dac9da9c72bc9116d.pdf"},{"id":99192235,"identity":"c4711092-fa82-4b1a-9b7e-6b954ec337e5","added_by":"auto","created_at":"2025-12-30 01:03:16","extension":"pdf","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29765,"visible":true,"origin":"","legend":"","description":"","filename":"Figure4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/bbb4ec84e30ba7ea783f7994.pdf"},{"id":99192239,"identity":"b81b7111-2883-4b3d-b651-136142e7fa1d","added_by":"auto","created_at":"2025-12-30 01:03:16","extension":"pdf","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":64565,"visible":true,"origin":"","legend":"","description":"","filename":"Figure5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/85eaeb828baa7309515c2d8c.pdf"},{"id":99317515,"identity":"9407808a-7fb0-4d3c-9c98-c4c8b0cbbb32","added_by":"auto","created_at":"2025-12-31 16:30:19","extension":"xml","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107972,"visible":true,"origin":"","legend":"","description":"","filename":"ERPCD25001650structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/5743286735e710bda729e253.xml"},{"id":99192240,"identity":"60a88b7d-496a-4f30-a0e3-d9b182db4316","added_by":"auto","created_at":"2025-12-30 01:03:16","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":118512,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/81f5b5f227e10f7ec37a5cf6.html"},{"id":99192225,"identity":"f7caab74-cb8d-4419-8d13-7fd1217cd750","added_by":"auto","created_at":"2025-12-30 01:03:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":16518,"visible":true,"origin":"","legend":"\u003cp\u003eRadiolabeling of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 Solution for Injection\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/bca8bdb297a238a704b1e993.png"},{"id":99192226,"identity":"1d35cb60-87ae-4ea1-bd8a-fa09027a9ce5","added_by":"auto","created_at":"2025-12-30 01:03:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":257856,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of Modular-Lab (E\u0026amp;Z) module used for the synthesis of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/5b749f21f9c421c53a6aaa79.png"},{"id":99192227,"identity":"9f8bc0b7-593d-428c-92e2-aaa7487f75e9","added_by":"auto","created_at":"2025-12-30 01:03:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":52676,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative HPLC chromatograms of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 at EOS.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/e9abcbc427d71cc2882ce96b.png"},{"id":99192232,"identity":"898a8574-fb6e-4f99-9ea7-ccaef649af6d","added_by":"auto","created_at":"2025-12-30 01:03:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30553,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative GC chromatograms of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/e6e5bc8e30a6b16de4b84ba2.png"},{"id":99317017,"identity":"14d76702-df1d-46cb-9857-6c856d521389","added_by":"auto","created_at":"2025-12-31 16:29:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":244772,"visible":true,"origin":"","legend":"\u003cp\u003e[\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 PET/CT of a hepatic metastatic lesion\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/d2b9f20e24b4626c13def5db.png"},{"id":99790582,"identity":"18d4545b-e122-4af0-9fd7-8e55906e0202","added_by":"auto","created_at":"2026-01-08 12:58:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1383859,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8384416/v1/9caef38e-688f-448f-ba2f-4a20c7b5a1c8.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eGMP-Compliant Manufacturing of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 for a Human Microdose Trial Targeting Platelet-Derived Growth Factor Receptor Beta (PDGFRβ)\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlatelet-derived growth factor receptor \u0026beta; (PDGFR\u0026beta;) is a transmembrane receptor tyrosine kinase that plays an important role in regulating various tissue functions, such as cell proliferation and extracellular matrix remodelling.\u0026nbsp;PDGFR\u0026beta; is activated upon binding of platelet-derived growth factor (PDGF) ligands, particularly PDGF-BB and PDGF-DD, which initiate intracellular signalling cascades that support mitogenic and anti-apoptotic responses as well as cytoskeletal reorganization.(1),(2)\u0026nbsp;Dysregulated PDGFR\u0026beta; signalling has been implicated in pathological processes such as fibrosis, atherosclerosis, and cancer.(3),(4)\u0026nbsp;In the liver, PDGFR\u0026beta; is crucial for fibrogenesis through the activation of hepatic stellate cells and the deposition of extracellular matrix, leading to fibrosis and cirrhosis.(5)\u0026nbsp;In liver tumours, such as metastases, PDGFR\u0026beta; is expressed by stromal cells in the tumour microenvironment\u0026nbsp;(6), and PDGFR\u0026beta;\u0026nbsp;inhibition synergized with chemotherapy in mouse models of colorectal cancer liver metastases\u0026nbsp;(7). Given its important role in both fibrosis and tumours, PDGFR\u0026beta; has emerged as an attractive therapeutic and diagnostic target. PDGFR\u0026beta;-directed monoclonal antibodies\u0026nbsp;(8)\u0026nbsp;have been developed and tested preclinically for antitumor activity,(9)\u0026nbsp;while several tyrosine kinase inhibitors (TKIs) (10)\u0026nbsp;with activity against PDGFR\u0026alpha;/\u0026beta; are already approved or under clinical investigation.(11)\u0026nbsp;Multi-target TKIs such as imatinib(12)\u0026nbsp;and sunitinib(13)\u0026nbsp;are approved in oncology, whereas nintedanib, a potent inhibitor of PDGFR\u0026alpha;/\u0026beta;, VEGFR, and FGFR has been approved for idiopathic pulmonary fibrosis and other progressive fibrotic lung diseases.(14)\u0026nbsp;These examples illustrate how PDGFR\u0026beta;-targeted strategies are being pursued in both malignant and non-malignant indications, highlighting its potential as an imaging biomarker and therapeutic target.(15)\u003c/p\u003e\n\u003cp\u003eHowever, patient stratification for PDGFR\u0026beta;-targeted therapies requires reliable detection of receptor expression. Conventional biopsy-based methods, such as immunohistochemistry, remain the clinical standard; however, they are invasive, limited by sample availability, and susceptible to false negatives due to inter- and intra-tumoral heterogeneity.(16) One promising strategy for non-invasive assessment of PDGFR\u0026beta; expression is molecular imaging, which can overcome the limitations of conventional biopsy-based methods by allowing dynamic, whole-body visualization of receptor expression and treatment response.(17) Among the available strategies developed for molecular imaging, Affibody molecules represent a new class of small, engineered scaffold proteins (6.5 kDa, three-helix bundle) with high thermal stability, rapid biodistribution, and excellent tissue penetration, making them well-suited for imaging applications compared to the larger antibodies.(18, 19) When used as PET tracers, Affibody molecules may significantly improve patient stratification and therapeutic monitoring, ultimately advancing precision medicine approaches targeting PDGFR\u0026beta;. Previous work has demonstrated the potential of PDGFR\u0026beta;-targeted Affibody molecules: Z09591, which binds human and murine PDGFR\u0026beta; with sub-nanomolar affinity, enabled high-contrast visualization of PDGFR\u0026beta;-expressing xenografts when labelled with \u003csup\u003e111\u003c/sup\u003eIn (20), \u003csup\u003e18\u003c/sup\u003eF (21, 22) and \u003csup\u003e68\u003c/sup\u003eGa (23). Based on these promising results, Z09591 was translated into a GMP-compliant format and re-designated as ATH001 for clinical development. The lead clinical candidate, [\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-Cys-ATH001 ([\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001), is labelled with gallium-68 for PET, enabling high-resolution whole-body imaging. Given the role of PDGFR\u0026beta; in fibrogenesis, we hypothesized that [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 could serve as a sensitive PET tracer for the detection and monitoring of liver fibrosis. This hypothesis is currently being investigated in an ongoing clinical trial (EU CT 2023-506555-15). In addition, since PDGFR\u0026beta;\u0026nbsp;is a recognized biomarker of tumour stroma, the tracer is also being evaluated in a separate ongoing study involving patients with primary and secondary liver tumours (EU CT 2025-522290-11-00). To ensure consistent and reliable production of high-quality tracers for molecular imaging in both trials, we developed a GMP-compliant manufacturing process for [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 using the ModularLab Pharmtracer synthesizer (Eckert \u0026amp; Ziegler). (24)\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eGeneral\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e[\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 was produced in a Class C environment using the GMP-compliant Modular-Lab PharmTracer synthesizer (Ecker \u0026amp; Ziegler). The synthesis module is located in a BBC-type hotcell (Comecer), with a product transfer line connecting it to a lead-shielded product vial hatch, allowing the product to be removed safely while minimizing operator radiation exposure. Both the hotcell and the product vial hatch maintained the same cleanroom classification as the surrounding laboratory environment. The preparation of the product vial was performed within a Class A laminar airflow microbiological safety cabinet (Ninolab), with a sterile product filter and a ventilation filter. The product vial is then transferred to the lead-shielded vial hatch, where the transfer line is attached to the inlet of the sterile product filter.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and equipment used for the synthesis of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DOTA-ATH001 peptide precursor (400 \u0026mu;g/vial), an Affibody molecule comprising a 59 amino acid sequence, was generated by Solid Phase Peptide Synthesis (in metal-free conditions) under full GMP compliance by Almac Sciences (Edinburgh, UK) for clinical radiolabelling. [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 was produced using a GMP-compliant Modular-Lab PharmTracer system (Eckert \u0026amp; Ziegler) with the corresponding reagent kit (EZ-102) and single-use hardware cassette (C4-GA-PEP), both specifically designed for the production of \u003csup\u003e68\u003c/sup\u003eGa-labeled peptides on the Modular-Lab PharmTracer platform. The synthesizer module was operated according to the manufacturer\u0026apos;s operation manual (Eckert \u0026amp; Ziegler, Berlin, Germany). The cassette and reagent kit were prepared in accordance with the manufacturer\u0026apos;s instructions. The Modular-Lab PharmTracer synthesis sequence was developed in-house at Karolinska Radiopharmacy, Karolinska University Hospital, based on the acetone-free \u003csup\u003e68\u003c/sup\u003eGa-DOTA peptide sequence provided by Eckert \u0026amp; Ziegler. The \u003csup\u003e68\u003c/sup\u003eGaCl\u003csub\u003e3\u003c/sub\u003e eluate was obtained from an Eckert \u0026amp; Ziegler 1.85 GBq \u003csup\u003e68\u003c/sup\u003eGe/\u003csup\u003e68\u003c/sup\u003eGa-generator (GalliaPharm, Eckert \u0026amp; Ziegler, Germany).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRadiolabelling of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe radiopharmaceutical [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 was synthesized using gallium-68 (\u003csup\u003e68\u003c/sup\u003eGa t\u003csub\u003e1/2\u003c/sub\u003e: 68 min) obtained as \u003csup\u003e68\u003c/sup\u003eGaCl\u003csub\u003e3\u003c/sub\u003e eluate from a\u0026nbsp;\u003csup\u003e68\u003c/sup\u003eGe/\u003csup\u003e68\u003c/sup\u003eGa-generator (Eckert \u0026amp; Ziegler). The generator was eluted by passing 5 mL of 0.1 N HCl into a sterile 15 mL glass vial (Huayi). The eluate activity was measured using a dose calibrator (Capintec) to determine the initial starting activity. The eluate was then transferred to the fully automated Modular-Lab PharmTracer synthesis module (Eckert \u0026amp; Ziegler) via syringe-driven transfer under control of the Modular-Lab software and connected to a single-use disposable cassette. Prior to radiolabelling, \u003csup\u003e68\u003c/sup\u003eGaCl\u003csub\u003e3\u0026nbsp;\u003c/sub\u003ewas concentrated on a cation exchange resin. Radiolabelling was performed by chelating gallium-68 with the DOTA moiety of ATH001 (200 \u0026mu;L, 1 mg/mL), previously mixed with EtOH/ TraceSELECT H\u003csub\u003e2\u003c/sub\u003eO 1:1 (0.4 mL),\u0026nbsp;buffer solution (0.4 mL; pH = 4) and TraceSELECT H\u003csub\u003e2\u003c/sub\u003eO (1 mL)\u0026nbsp;at 65 \u0026deg;C for 10 minutes, yielding a specific activity (A\u003csub\u003es\u003c/sub\u003e) \u0026ge; 160 MBq/mg. The crude\u0026nbsp;[\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 reaction mixture was subsequently purified by solid-phase extraction (HLB light SPE cartridge Oasis, Waters), eluted with 50% ethanol, and formulated in sterile saline (0.9%). The final product solution was sterilized by filtration through a 0.22 \u0026mu;m sterile filter (Millex-GV 0.22 \u0026micro;m,\u0026nbsp;Merck Millipore).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEquipment used for the quality control of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHigh-performance liquid chromatography (HPLC) was performed using an Agilent 1260 Infinity system (Agilent Technologies) equipped with a G7111B pump, G7129A vial sampler, and a G7130A integrated column compartment. The system was coupled to a VWR detector (G7114A) and a radio-detector Flow-RAM with a 1\u0026Prime; NaI PMT (LabLogic) and connected using blue tubing (0.25 mm, five wraps). Data acquisition and evaluation were carried out with Laura software (LabLogic). The analytical column used was a Poroshell 120 EC-C18 column (3 \u0026times; 150 mm, 2.7 \u0026mu;m) protected by a Poroshell 120 EC-C18 fast guard column (3 \u0026times; 5 mm, 2.7 \u0026mu;m), with a column volume of 1 mL. The mobile phases consisted of (A) 0.1% trifluoroacetic acid (TFA) in water and (B) 60% acetonitrile (ACN) in 0.1% TFA. The applied gradient method (Ga-ATH001-HPLC) started with 60% A and 40% B, which was held for 1.5 min, followed by a linear increase to 100% B over 9 min, maintained for 2 min, and returned to the initial conditions for re-equilibration, giving a total run time of 15 min. The flow rate was set at 0.4 mL/min, the column temperature was maintained at room temperature, and the injection volume was 50 \u0026mu;L. Seal wash was performed with 20% ethanol in Milli-Q water. UV detection was carried out at 220 nm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe pH of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 was determined using pH indicator strips 2.0-9.0 (VWR) or pH-meter (type 913, version 2.913.0210, Metrohm).\u003c/p\u003e\n\u003cp\u003eThe integrity of the filter was assessed using a bubble point test with custom-made equipment (010105280602-A, DM Automation).\u003c/p\u003e\n\u003cp\u003eGas chromatography (GC) was carried out on an Agilent 6850 system equipped with a flame ionization detector (FID) and controlled by Laura 6 software (LabLogic). A Res-Solv column (30 m \u0026times; 0.53 mm i.d., 1.0 \u0026mu;m film) with an autoinjector was used. The split ratio was set to 1:80, and the injection volume was 2 \u0026mu;L. Helium served as the carrier gas, while hydrogen and synthetic air were used for the detector. The inlet temperature was maintained at 250 \u0026deg;C with an inlet pressure of 2.76 PSI. The oven temperature program was as follows: initial isotherm at 35 \u0026deg;C for 4 min, ramp to 100 \u0026deg;C at 80 \u0026deg;C/min and hold for 30 s, ramp to 220 \u0026deg;C at 80 \u0026deg;C/min and hold for 30 s, followed by cooling to 35 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003eThe specific activity was a critical parameter for this product, defined as the ratio of the radioactivity of [⁶⁸Ga]Ga-ATH001 to the total mass of DOTA-Cys-ATH001 (ATH001) as described in Equation 1. It was expressed in GBq/mg and determined both at the end of synthesis (EOS) and at EOS plus shelf-life (60\u0026ndash;90 minutes post-synthesis), see Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003eThe radioactivity of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 is measured in a dose calibrator. The mass of DOTA-Cys-ATH001 is determined by liquid chromatography with UV detection and comparison of the area counts from the product sample with those from a known amount of ATH001 precursor (using a linearity function).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePET imaging in human\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA phase one study is currently ongoing and includes a cohort of participants with liver metastases and intrahepatic cholangiocarcinoma (EU CT 2025-522290-11-00). No definitive or preliminary data are available yet, as recruitment has only just begun. However, a representative administered activity was estimated at 200 MBq \u0026plusmn; 10% (approximately 2.5 MBq/kg body weight). The scanning session began with a dynamic scan of the liver for about 45 minutes after injection of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001. This was followed by whole-body static PET acquisitions at 60 minutes and a static scan over the liver at 135 minutes post-injection. The radiation exposure from the administered activity was below 6 mSv. Non-contrast CT scans are used for attenuation correction and anatomical co-registration of PET images, resulting in a total radiation exposure (PET and CT components) of around 13.5 mSv.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe radiolabelling of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001, illustrated in Fig.\u0026nbsp;1, was performed under metal-free conditions using a GMP-compliant automated synthesizer. The in-house developed synthesis sequence for [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 Solution for Injection involved three main steps:\u003c/p\u003e \u003cp\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Trapping and pre-concentration of [\u003csup\u003e68\u003c/sup\u003eGa]Ga\u003csup\u003e3+\u003c/sup\u003e; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) Chelation of [\u003csup\u003e68\u003c/sup\u003eGa]Ga\u003csup\u003e3+\u003c/sup\u003e with the DOTA moiety of ATH001 precursor; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) Purification, formulation and sterile filtration of the final product. The synthesis was performed in the following sequences in Fig.\u0026nbsp;2:\u003c/p\u003e \u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe \u003csup\u003e68\u003c/sup\u003eGaCl\u003csub\u003e3\u003c/sub\u003e eluate collected in a sterile vial from the generator was withdrawn by the module and loaded onto a strong cationic exchange (SCX) cartridge. [\u003csup\u003e68\u003c/sup\u003eGa]Ga\u003csup\u003e3+\u003c/sup\u003e was retained on the cartridge, while potential \u003csup\u003e68\u003c/sup\u003eGe contaminants were eluted to the waste. The retained \u003csup\u003e68\u003c/sup\u003eGa was subsequently released into the reaction vessel of the cassette by eluting with 0.7 mL of 5M NaCl/0.13N HCl (pH\u0026thinsp;\u0026lt;\u0026thinsp;2).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe radiolabelling step was performed by complexation of [\u003csup\u003e68\u003c/sup\u003eGa]Ga\u003csup\u003e3+\u003c/sup\u003e with the DOTA chelator of the precursor DOTA-Cys-ATH001 (V\u0026thinsp;=\u0026thinsp;200 \u0026micro;L, C\u0026thinsp;=\u0026thinsp;1 mg/mL), in the reaction vessel containing a EtOH/H\u003csub\u003e2\u003c/sub\u003eO 1:1 mixture (V\u0026thinsp;=\u0026thinsp;0.4 mL), buffer solution (V\u0026thinsp;=\u0026thinsp;0.4 mL of sodium acetate pH\u0026thinsp;=\u0026thinsp;4) prepared from the EZ-102 reagent kit according to the Eckert \u0026amp; Ziegler \u0026ldquo;user manual for synthesis of [\u003csup\u003e68\u003c/sup\u003eGa]-conjugated peptides with PharmTracer fractionation and pre-purifications by cation exchange\u0026rdquo; and TraceSELECT H\u003csub\u003e2\u003c/sub\u003eO (V\u0026thinsp;=\u0026thinsp;1 mL). The reaction mixture had a pH of 3.5-4 and was heated at 65\u0026deg;C for 10 min (Fig.\u0026nbsp;1).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe crude [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 reaction mixture was diluted with sterile saline (9 mg/mL, 5 mL) prior to purification using a reversed phase solid phase extraction (SPE) cartridge (Oasis light HLB SPE within the EZ cassette). The product was eluted with an EtOH/H\u003csub\u003e2\u003c/sub\u003eO 1:1 mixture and formulated with sterile sodium chloride (9 mg/mL, reagent kit EZ-102). The final product was sterilized through a 0.22 \u0026micro;m sterile membrane filter and collected in a 15 mL sterile vial (Huayi) as [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 Solution for Injection.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e \u003cp\u003eFigure 1. Radiolabeling of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 Solution for Injection\u003c/p\u003e \u003cp\u003eFigure 2. Flowchart of Modular-Lab (E\u0026amp;Z) module used for the synthesis of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001\u003c/p\u003e \u003cp\u003eAn automated GMP-compliant labelling process for [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 was successfully implemented in which, three consecutive batches were produced under normal conditions and one batch was performed to control potential microbiological burden originated from the radio-synthesizer, yielding an average decay-corrected radiochemical yield of 49.8\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6% (n\u0026thinsp;=\u0026thinsp;4), with an average final product activity of 429.3\u0026thinsp;\u0026plusmn;\u0026thinsp;27.4 MBq (n\u0026thinsp;=\u0026thinsp;4) and a radiochemical purity of 96.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4% among all batches. The mean specific activity at the estimated administration time (shelf-life included) was 2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 GBq/mg (n\u0026thinsp;=\u0026thinsp;4). Stability studies have been performed where the radiochemical stability of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 was 96.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4% after 2 hours at room temperature (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\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\u003eProcess validation of [68Ga]Ga-ATH001\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProduct specification\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBatch 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBatch 2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBatch 3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBatch 4\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eActivity product at EOS in MBq (after withdrawal of QC and sterility samples)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;160 MBq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e452 MBq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e431 MBq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e450 MBq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e384 MBq\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVolume of product in mL (after withdrawal of QC and sterility samples)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;10 mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.28 mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.24 mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.14 mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.65\u003csup\u003eb\u003c/sup\u003e mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eActivity concentration (MBq/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e62.05 MBq/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e59.49 MBq/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63.06 MBq/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50.19 MBq/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAppearance\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClear, colourless or slightly yellow. Free of visual particles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eComply\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eComply\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eComply\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eComply\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\u003e4.0\u0026ndash;8.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadionuclidic identity half-life \u003csup\u003e68\u003c/sup\u003eGa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62 to 74 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e69 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e68 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e70 min\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConcentration of ATH001\u003c/p\u003e \u003cp\u003e(principal peak on SST UV)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u0026ndash;20 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.97 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.87\u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.3 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.91 \u0026micro;g/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChemical impurity(ies)\u003c/p\u003e \u003cp\u003e(peaks except principal peak detected on UV)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u0026ndash;5%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProduct identification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e[Rt\u003csub\u003eRD\u003c/sub\u003e\u0026ndash; Rt\u003csub\u003eUV\u003c/sub\u003e| \u0026le; 60 s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6 s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8 s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8 s\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11 s\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadiochemical purity HPLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;91%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e97%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e97%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e96%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecific activity EOS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;1.6 GBq/mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.44 GBq/mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.76 GBq/mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.88 GBq/mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.91 GBq/mg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecific activity (EOS\u0026thinsp;+\u0026thinsp;shelf-life)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;1.6 GBq/mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.28 GBq/mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.51 GBq/mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.45 GBq/mg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.46 GBq/mg\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFilter integrity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;3.5 bar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.2 bar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.1 bar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.2 bar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN.A\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSterility\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 \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSterile\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBacterial endotoxins\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;5.00 IU/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5.00 IU/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5.00 IU/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5.00 IU/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthanol content\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026le;\u0026thinsp;80 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e50 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e44 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e47 mg/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadiochemical Stability\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRCP\u0026thinsp;\u0026ge;\u0026thinsp;91% after 120 minutes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e97%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e96%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003eAbbreviations: Rt\u003csub\u003eRD\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;retention time from radio-detector; Rt\u003csub\u003eUV\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;retention time from UV detector, EOS\u0026thinsp;=\u0026thinsp;end of synthesis.\u003c/p\u003e \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Microbiological worst-case challenge: a batch in which the sterile filter is intentionally excluded.\u003c/p\u003e \u003cp\u003e\u003csup\u003eb\u003c/sup\u003e A larger product volume is observed due to the omission of the sterile filter.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eQuality control\u003c/h3\u003e\n\u003cp\u003eValidation of the analytical methods confirmed that the procedures are reliable and appropriate for their intended purpose. The results of quality control testing of all product validation batches are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Representative chromatograms from HPLC, and GC analyses are presented in Figs.\u0026nbsp;3, and 4, respectively. The HPLC chromatogram of [⁶⁸Ga]Ga-ATH001 (Fig.\u0026nbsp;3) presents a dominant peak at Rt\u0026thinsp;\u0026asymp;\u0026thinsp;8.53 min as well as the UV peak of the non-radioactive precursor (UV Rt\u0026thinsp;\u0026asymp;\u0026thinsp;8.45 min). [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 has an RCP of ~\u0026thinsp;96.8%, with traces of \u003csup\u003e68\u003c/sup\u003eGa-ions (RCP\u0026thinsp;\u0026le;\u0026thinsp;1.3% within the region of interest (ROI). The total RCP of \u003csup\u003e68\u003c/sup\u003eGa-related impurities was \u0026le;\u0026thinsp;1.8%, which is consistent with the specifications (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The UV peak corresponding to ATH001 had an integrated area with a concentration of 11.25 \u0026micro;g\u0026middot;mL⁻\u0026sup1;, as determined from the previously established calibration curve. This concentration was used to calculate the injected molar amount and the specific activity, which is a critical parameter for the planned microdose study.\u003c/p\u003e \u003cp\u003eFigure 3. Representative HPLC chromatograms of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 at EOS.\u003c/p\u003e \u003cp\u003e[\u003csup\u003e68\u003c/sup\u003eGa]Ga\u003csup\u003e3+\u003c/sup\u003e ions and other radiochemical impurities may be retained in the injection system, tubing or the column material itself. Several approaches exist to assess these retention effects and must be considered during method validation. To address this, we compared the injected radioactivity with that recovered in the eluent. This can be achieved by collecting the eluent in fractions and measuring their radioactivity, then comparing these values with the injected activity.(\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eTo omit the additional iTLC procedure, which is time-consuming and may negatively impact the specific activity of the final product due to time delay for batch release, a column recovery has been performed where the radioactivity of the QC sample (200 \u0026micro;L) was first measured with a Capintec dose calibrator, applying appropriate decay time correction. Subsequently, 50 \u0026micro;L of the product was injected onto the HPLC column. During the 15-minute HPLC run, all eluate from the column was collected, and its radioactivity measured using the dose calibrator. The residual activity remaining in the HPLC injection vial was also recorded. By comparing the time-corrected total activity recovered from the column eluate plus the residual vial activity to the initial activity in the product vial, the recovery percentage was calculated and found to be in the range of 90\u0026ndash;110%, confirming near-complete elution of the radiolabelled compound.\u003c/p\u003e \u003cp\u003eThe HPLC recovery is calculated following the Eq.\u0026nbsp;2 in-below:\u003c/p\u003e \u003cp\u003e \u003cb\u003eEquation 2\u003c/b\u003e: \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:Recovery\\:\\left(\\%\\right)=\\frac{\\text{A}\\text{c}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}\\:\\text{l}\\text{e}\\text{f}\\text{t}\\:\\text{i}\\text{n}\\:\\text{v}\\text{i}\\text{a}\\text{l}\\:\\text{a}\\text{f}\\text{t}\\text{e}\\text{r}\\:\\text{i}\\text{n}\\text{j}\\text{e}\\text{c}\\text{t}\\text{i}\\text{o}\\text{n}+\\text{A}\\text{c}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}\\:\\text{c}\\text{o}\\text{l}\\text{l}\\text{e}\\text{c}\\text{t}\\text{e}\\text{d}\\:\\text{f}\\text{r}\\text{o}\\text{m}\\:\\text{c}\\text{o}\\text{l}\\text{u}\\text{m}\\text{n}}{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{a}\\text{c}\\text{t}\\text{i}\\text{v}\\text{i}\\text{t}\\text{y}\\:\\text{i}\\text{n}\\:\\text{v}\\text{i}\\text{a}\\text{l}\\:\\text{b}\\text{e}\\text{f}\\text{o}\\text{r}\\text{e}\\:\\text{i}\\text{n}\\text{j}\\text{e}\\text{c}\\text{t}\\text{i}\\text{o}\\text{n}}*100\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003eThe GC analysis of the final product showed a single ethanol peak (Rt\u0026thinsp;~\u0026thinsp;2.5min) and only trace/LLQ signal for acetone (Fig.\u0026nbsp;4). Quantification of the ethanol calibration curve gave an ethanol content that met our predefined release limit and is compliant with Class-3 solvent expectations (ICH Q3C). No additional volatile impurities were detected above the reporting threshold. Therefore, the ethanol level of the formulation is within acceptable limits for clinical use and aligns with its role as a stabilizer agent.\u003c/p\u003e \u003cp\u003eFigure 4. Representative GC chromatograms of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAn automated GMP-compliant process for [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 was successfully implemented on the ModularLab synthesis module, with high radiochemical purity 96.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4% (n\u0026thinsp;=\u0026thinsp;4) and high specific activity (2.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1 GBq/mg at the time of administration), in compliance with microdosing requirements. Stability tests of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 demonstrated that the radiochemical purity remained\u0026thinsp;\u0026ge;\u0026thinsp;96% for up to 2 hours at room temperature with no trace of radiolysis, thus eliminating the need for additional stabilizers. These results are comparable to the automated process developed by Jussing et al. for a different Affibody molecule-based PET tracer [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ABY-025,(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) where validation batches achieved high yields, purity, and stability while using ModularLab synthesis module, including a worst-case batch without sterile filtration to study microbiological robustness. These findings demonstrate that the automated \u003csup\u003e68\u003c/sup\u003eGa-labeling process is both reliable and applicable to translational and clinical use.\u003c/p\u003e \u003cp\u003eFollowing the findings on [\u003csup\u003e68\u003c/sup\u003eGa]DOTA-Z09591 (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), our fully automated process provides harmonization, GMP reproducibility, and optimized precursor use achieving high specific activity without radiolysis. In contrast, \u003csup\u003e18\u003c/sup\u003eF-labeling strategies such as [\u003csup\u003e18\u003c/sup\u003eF]TZ-Z09591(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) require two-steps approach, involving radiosynthesis of a prosthetic tetrazine group followed by a TCO-tetrazine click chemistry ligation to the Affibody molecule. While efficient, this resulted in moderate molar activity (~\u0026thinsp;20 MBq/nmol) and increased operational complexity. Thus, although \u003csup\u003e18\u003c/sup\u003eF provides advantages due to its longer half-life and the possibility of large-scale production, the use of \u003csup\u003e68\u003c/sup\u003eGa is particularly attractive for clinical studies because of its on-site generator availability, well-established clinical use, and straightforward DOTA chemistry.(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e) Moreover, alternative chelation strategies, such as RESCA for Al-\u003csup\u003e18\u003c/sup\u003eF labelling, have shown inferior in vivo stability compared to DOTA or NOTA, with the evidence of Ga-RESCA trans-chelation under serum and acidic conditions. This may reduce imaging clarity and increase off-target radiation.(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) This reduced robustness, combined with more limited clinical validation, makes \u003csup\u003e68\u003c/sup\u003eGa-DOTA chemistry the more reliable choice for early clinical development.(\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe selection of PDGFRβ as the biological target for an imaging agent offers various advantages over alternative strategies such as fibroblast activation protein (FAP), another emerging PET-compatible stromal marker. While FAP-targeted tracers are of strong interest for their high tumour-to-background ratios, particularly in desmoplastic tumours,(\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e) PDGFRβ represents a more direct and clinically validated target. Several PDGFRβ inhibitors, including imatinib(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), sunitinib(\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and pazopanib, are FDA-approved and used in oncology. Moreover, PDGFRβ itself is frequently expressed on tumour cells and pericytes, as well as cancer-associated fibroblasts in the tumour microenvironment, potentially enabling direct visualization of tumour biology and angiogenesis rather than solely stromal components alone.(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) Furthermore, PDGFR signalling has been extensively studied in oncogenesis, fibrosis, and angiogenesis, providing strong mechanistic support for imaging applications, with isoform-specific biology (PDGFRα vs PDGFRβ) allowing additional precision. Despite potential limitations such as physiological expression in normal tissues and the risk of resistance mechanisms, PDGFRβ remains a well-validated therapeutic target, potentially providing a more direct translational pathway than FAP for early diagnostic development.\u003c/p\u003e \u003cp\u003eThe optimized automated production of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 on a ModularLab synthesizer, with a RCY of \u0026ge;\u0026thinsp;49.8% (n\u0026thinsp;=\u0026thinsp;4) and a RCP of \u0026ge;\u0026thinsp;96.8%, provide a high-quality tracer with excellent reproducibility in compliance with GMP specifications for a clinical trial. Further improvements could include the use of cyclotron-produced \u003csup\u003e68\u003c/sup\u003eGa to achieve higher specific activity of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001. Recent advances in solid-target cyclotron production with optimized purification methods (e.g., ascorbate-assisted UTEVA resin processing) have enabled apparent molar activities approaching 500 GBq/\u0026micro;mol and batch yields up to 7 GBq, representing a ten-fold increase compared to generator-based production.(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e) Such developments could facilitate large-scale multi-center trials and broaden clinical accessibility. This positions [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 as a clinically translatable PET tracer that not only builds on prior experience with \u003csup\u003e68\u003c/sup\u003eGa-labeled Affibody molecules, (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) but also overcomes limitations of \u003csup\u003e18\u003c/sup\u003eF-based approaches (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) and provides advantages over emerging alternatives such as FAPI.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eClinical PET/CT scanning\u003c/h2\u003e \u003cp\u003eThe [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 PET/CT examination was performed on a GE Discovery DMI generation 2 (Milwaukee, WI, USA) scanner. Representative data from PET/CT images of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 in a study participant with a liver metastasis from colorectal cancer are shown in Fig.\u0026nbsp;5. The liver metastasis exhibited increased tracer uptake compared with the surrounding healthy liver tissue, even at early time points.\u003c/p\u003e \u003cp\u003eFigure 5. [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 PET/CT of a hepatic metastatic lesion\u003c/p\u003e \u003cp\u003eRepresentative images from a study participant with a large metastatic lesion in liver segments 6/7 are shown on contrast-enhanced axial CT (Fig.\u0026nbsp;5a). [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 PET/CT examination reveals increased tracer uptake (arrows) compared with the surrounding liver parenchyma at both early (Fig.\u0026nbsp;5b,c) and late (Fig.\u0026nbsp;5d,e) time points.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe GMP-compliant automated synthesis of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 consistently delivers a high-quality PET tracer suitable for clinical use. This advancement enables non-invasive characterization of PDGFRβ expression in tissue and supports progress in both cancer- and fibrosis-related research, as well as anti-fibrotic drug development. Further improvements could include the use of cyclotron-produced \u003csup\u003e68\u003c/sup\u003eGa to increase batch activity, enhance production scalability, and allow imaging of multiple participants from a single synthesis of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001. The ongoing IMAGINE-1 trial will evaluate PDGFRβ-PET for visualization of tumour stroma and angiogenesis in participants with liver tumours and intrahepatic cholangiocarcinoma, expanding the clinical applications of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 across oncologic and fibrotic indications.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- ACN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcetonitrile\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- Al-\u003csup\u003e18\u003c/sup\u003eF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAluminium-[\u003csup\u003e18\u003c/sup\u003eF]Fluoride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- As\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSpecific activity (GBq/mg)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- cGMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCurrent Good Manufacturing Practices\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- CT\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\"\u003e- DOTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- EOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnd of Synthesis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- EtOH\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\"\u003e- E\u0026amp;Z\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEckert \u0026amp; Ziegler\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- FAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFibroblast activation protein\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- FGFR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFibroblast Growth Factor Receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- FID\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFlame ionization detector\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e[\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-Cys-ATH001\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- GC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGas chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- GMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGood manufacture practices\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- H\u003csub\u003e2\u003c/sub\u003eO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWater\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- HCl\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\"\u003e- HLB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHydrophilic-lipophilic balance cartridge\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- HPLC\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\"\u003e- IMAGINE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eImatinib Augmented Perfusion for GastroIntestinal Liver Neoplasia\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- iTLC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einstant Thin layer chromatography\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- NaCl\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSodium chloride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- NOTA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e1,4,7-triazacyclononane-1,4,7-triacetic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- PET\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\"\u003e- PDGFRβ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePlatelet-derived growth factor receptor beta\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- PSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePound-force per square inch \u0026ndash; Unit of pressure\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- QC\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\"\u003e- RCP\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\"\u003e- RCP\u003csub\u003eTot\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTotal radiochemical purity\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- RCY\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\"\u003e- RD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRadio-detector\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- RESCA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRapid, Efficient and Stable chelator for Aluminium-[\u003csup\u003e18\u003c/sup\u003eF]Fluoride\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- ROI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRegion of interest\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- Rt\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRetention time\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- Rt\u003csub\u003eRD\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRetention time from radio-detector\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- Rt\u003csub\u003eUV\u003c/sub\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRetention time from UV detector\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- SCX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eStrong cationic exchange cartridge\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- TFA\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\"\u003e- TKI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTyrosine kinase inhibitors\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- UV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUltra-violet\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e- VEGFR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVascular Endothelial Growth Factor Receptor\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe clinical PET imaging study was approved by the Swedish Ethical Review Authority under EU CT 2025-522290-11-00,and all participants gave written consent before initiating any study procedure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe clinical patient data used in the publication have been anonymized and safeguards measures taken to follow the GDPR (General Data Protection Regulation) requirements according to the Karolinska University Hospital\u0026rsquo;s regulation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSummary data supporting the findings of the current study are included within the article. Raw PET imaging data are not publicly available due to ethical and participant privacy considerations but may be available from the corresponding author upon reasonable request and subject to appropriate approval. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe GMP validation was funded by Antaros Tracer AB. The precursor materials were supplied by Antaros Tracer AB, under license from Affibody AB. The First-in-Human microdose study (2023-506555-15) was funded by Takeda Pharmaceutical Company Limited and Antaros Tracer AB, whereas the clinical program (2025-522290-11-00) described in this paper is supported by funding from Radiumhemmet\u0026acute;s Research Fund, Karolinska Institutet\u0026rsquo;s Microenvironmental Control of Metastasis network, and a private donor.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAyman Abouzayed, Per Hagmar and Olof Eriksson are employees at Antaros Tracer AB. Talakad Lohith is an employee at Takeda Pharmaceuticals Company Limited. Olof Eriksson is mentioned as inventor of a patent relating to PDGFR\u0026beta; binding peptides. The remaining authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. M\u0026eacute;lodie Ferrat, Mohammad Mahdi Moein, Tetyana Tegnebratt, Emma Jussing and Thuy Tran were responsible for the GMP validation and manufacturing of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001. Ayman Abouzayed, Per Hagmar and Olof Eriksson designed and developed the peptide precursor. M\u0026eacute;lodie Ferrat developed the radiolabelling procedure in the GMP laboratory and was the major contributor in writing the manuscript. Rimma Axelsson, Antonios Tzorzakakis, Marco Gerling and Jennie Engstrand, contributed with expertise regarding the clinical examination for which the radiotracer production is intended. All authors contributed to reading and approving the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all members part of this study for their help and contribution as well as Karolinska Radiopharmacy group for their support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eFriedman SL, Sheppard D, Duffield JS, Violette S. Therapy for Fibrotic Diseases: Nearing the Starting Line. Science translational medicine. 2013;5(167):167sr1.\u003c/li\u003e\n \u003cli\u003eDobie R, Connelly J, Henderson NC. PDGF-Mediated Regulation of Liver Fibrosis. Current pathobiology reports. 2015;3(4):225\u0026ndash;33.\u003c/li\u003e\n \u003cli\u003eHeldin C-H. Targeting the PDGF Signaling Pathway in the Treatment of Non-Malignant Diseases. Journal of neuroimmune pharmacology. 2014;9(2):69\u0026ndash;79.\u003c/li\u003e\n \u003cli\u003eHeldin C-H. Targeting the PDGF signaling pathway in tumor treatment. Cell communication and signaling. 2013;11(1):97\u0026ndash;.\u003c/li\u003e\n \u003cli\u003eHeldin CH, Westermark B. Platelet-derived growth factor: mechanism of action and possible in vivo function. Cell regulation. 1990;1(8):555\u0026ndash;66.\u003c/li\u003e\n \u003cli\u003eFern\u0026aacute;ndez Moro C, Geyer N, Harrizi S, Hamidi Y, S\u0026ouml;derqvist S, Kuznyecov D, et al. An idiosyncratic zonated stroma encapsulates desmoplastic liver metastases and originates from injured liver. Nat Commun. 2023;14(1):5024.\u003c/li\u003e\n \u003cli\u003eKitadai Y, Sasaki T, Kuwai T, Nakamura T, Bucana CD, Fidler IJ. Targeting the expression of platelet-derived growth factor receptor by reactive stroma inhibits growth and metastasis of human colon carcinoma. Am J Pathol. 2006;169(6):2054\u0026ndash;65.\u003c/li\u003e\n \u003cli\u003eOgawa S, Ochi T, Shimada H, Inagaki K, Fujita I, Nii A, et al. Anti-PDGF-B monoclonal antibody reduces liver fibrosis development. Hepatology research. 2010;40(11):1128\u0026ndash;41.\u003c/li\u003e\n \u003cli\u003eShen J, Vil MD, Prewett M, Damoci C, Zhang H, Li H, et al. Development of a fully human anti-PDGFRbeta antibody that suppresses growth of human tumor xenografts and enhances antitumor activity of an anti-VEGFR2 antibody. Neoplasia. 2009;11(6):594\u0026ndash;604.\u003c/li\u003e\n \u003cli\u003eStacchiotti S, Longhi A, Ferraresi V, Grignani G, Comandone A, Stupp R, et al. Phase II Study of Imatinib in Advanced Chordoma. Journal of clinical oncology. 2012;30(9):914\u0026ndash;20.\u003c/li\u003e\n \u003cli\u003eDai Y. Platelet-derived growth factor receptor tyrosine kinase inhibitors: a review of the recent patent literature. Expert Opin Ther Pat. 2010;20(7):885\u0026ndash;97.\u003c/li\u003e\n \u003cli\u003eFDA. Drug approval: Gleevec (Imatinib Mesylate) Capsules 2001 [Available from: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2001/21335_Gleevec.cfm#:~:text=Approval%20Date:%205/10/,Part%202%20(PDF).\u003c/li\u003e\n \u003cli\u003e(FDA) USFaDA. Drug Approval: Sutent (Sunitinib Malate) Capsules 2006 [Available from: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2006/021938s000_\u003cbr\u003e021968s000_Stutent.cfm#:~:text=Approval%20Date:%2001/26/,s)%20\u0026amp;%20\u003cbr\u003eCorrespondence%20(PDF).\u003c/li\u003e\n \u003cli\u003eFala L. Ofev (Nintedanib): First Tyrosine Kinase Inhibitor Approved for the Treatment of Patients with Idiopathic Pulmonary Fibrosis. Am Health Drug Benefits. 2015;8(Spec Feature):101\u0026ndash;4.\u003c/li\u003e\n \u003cli\u003eYing HZ, Chen Q, Zhang WY, Zhang HH, Ma Y, Zhang SZ, et al. PDGF signaling pathway in hepatic fibrosis pathogenesis and therapeutics (Review). Mol Med Rep. 2017;16(6):7879\u0026ndash;89.\u003c/li\u003e\n \u003cli\u003eFriedman SL, Pinzani M. Hepatic fibrosis 2022: Unmet needs and a blueprint for the future. Hepatology (Baltimore, Md). 2022;75(2):473\u0026ndash;88.\u003c/li\u003e\n \u003cli\u003eTolmachev V, Stone-Elander S, Orlova A. Radiolabelled receptor-tyrosine-kinase targeting drugs for patient stratification and monitoring of therapy response: prospects and pitfalls. The lancet oncology. 2010;11(10):992\u0026ndash;1000.\u003c/li\u003e\n \u003cli\u003eL\u0026ouml;fblom J, Feldwisch J, Tolmachev V, Carlsson J, St\u0026aring;hl S, Frejd FY. Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS letters. 2010;584(12):2670\u0026ndash;80.\u003c/li\u003e\n \u003cli\u003eLindborg M, Cortez E, H\u0026ouml;id\u0026eacute;n-Guthenberg I, Gunneriusson E, von Hage E, Syud F, et al. Engineered High-Affinity Affibody Molecules Targeting Platelet-Derived Growth Factor Receptor \u0026beta; In Vivo. Journal of molecular biology. 2011;407(2):298\u0026ndash;315.\u003c/li\u003e\n \u003cli\u003eTolmachev V, Varasteh Z, Honarvar H, Hosseinimehr SJ, Eriksson O, Jonasson P, et al. Imaging of platelet-derived growth factor receptor \u0026beta; expression in glioblastoma xenografts using affibody molecule 111In-DOTA-Z09591. The Journal of nuclear medicine (1978). 2014;55(2):294.\u003c/li\u003e\n \u003cli\u003eWegrzyniak O, Zhang B, Rokka J, Rosestedt M, Mitran B, Cheung P, et al. Imaging of fibrogenesis in the liver by [18F]TZ-Z09591, an Affibody molecule targeting platelet derived growth factor receptor \u0026beta;. EJNMMI radiopharmacy and chemistry. 2023;8(1):23\u0026ndash;18.\u003c/li\u003e\n \u003cli\u003eWegrzyniak O, Lechi F, Mitran B, Cheung P, Bitzios A, Persson J, et al. Non-invasive PET imaging of liver fibrogenesis using a RESCA-conjugated Affibody molecule. iScience. 2024;27(5):109688.\u003c/li\u003e\n \u003cli\u003eStrand J, Varasteh Z, Eriksson O, Abrahmsen L, Orlova A, Tolmachev V. Gallium-68-Labeled Affibody Molecule for PET Imaging of PDGFR\u0026beta; Expression in Vivo. Molecular pharmaceutics. 2014;11(11):3957\u0026ndash;64.\u003c/li\u003e\n \u003cli\u003eLubberink M, editor Dosimetry and safety of the PDGFRb PET tracer [68Ga]GaDOTA-Cys-ATH001: first-in-human data in patients with liver fibrosis and healthy subjects. Annual Congress of the European Association of Nuclear Medicine (EANM) 2025; 2025; Barcelona, Spain: Elsevier.\u003c/li\u003e\n \u003cli\u003eGillings N, Todde S, Behe M, Decristoforo C, Elsinga P, Ferrari V, et al. EANM guideline on the validation of analytical methods for radiopharmaceuticals. EJNMMI Radiopharmacy and Chemistry. 2020;5(1):7.\u003c/li\u003e\n \u003cli\u003eJussing E, Ferrat M, Moein MM, Alfred\u0026eacute;en H, Tegnebratt T, Bratteby K, et al. Optimized, automated and cGMP-compliant synthesis of the HER2 targeting [68Ga]Ga-ABY-025 tracer. EJNMMI radiopharmacy and chemistry. 2023;8(1):41\u0026ndash;18.\u003c/li\u003e\n \u003cli\u003eFani M, Andr\u0026eacute; JP, Maecke HR. 68Ga-PET: a powerful generator-based alternative to cyclotron-based PET radiopharmaceuticals. Contrast media and molecular imaging. 2008;3(2):53\u0026ndash;63.\u003c/li\u003e\n \u003cli\u003eLindner T, Loktev A, Altmann A, Giesel F, Kratochwil C, Debus J, et al. Development of Quinoline-Based Theranostic Ligands for the Targeting of Fibroblast Activation Protein. Journal of Nuclear Medicine. 2018;59(9):1415\u0026ndash;22.\u003c/li\u003e\n \u003cli\u003eKratochwil C, Flechsig P, Lindner T, Abderrahim L, Altmann A, Mier W, et al. 68 Ga-FAPI PET/CT: Tracer Uptake in 28 Different Kinds of Cancer. Journal of Nuclear Medicine. 2019;60(6):801\u0026ndash;5.\u003c/li\u003e\n \u003cli\u003eDemetri GD, von Mehren M, Blanke CD, Van den Abbeele AD, Eisenberg B, Roberts PJ, et al. Efficacy and Safety of Imatinib Mesylate in Advanced Gastrointestinal Stromal Tumors. The New England journal of medicine. 2002;347(7):472\u0026ndash;80.\u003c/li\u003e\n \u003cli\u003eMotzer RJ, Hutson TE, Tomczak P, Michaelson MD, Bukowski RM, Rixe O, et al. Sunitinib versus Interferon Alfa in Metastatic Renal-Cell Carcinoma. The New England journal of medicine. 2007;356(2):115\u0026ndash;24.\u003c/li\u003e\n \u003cli\u003eJussing E, Milton S, Sam\u0026eacute;n E, Moein MM, Bylund L, Axelsson R, et al. Clinically Applicable Cyclotron-Produced Gallium-68 Gives High-Yield Radiolabeling of DOTA-Based Tracers. Biomolecules. 2021;11(8):1118.\u003c/li\u003e\n\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":"Positron Emission Tomography (PET), [68Ga]Ga-ATH001, Radiopharmaceuticals, Platelet-Derived Growth Factor Receptor Beta (PDGFRβ), Fibrogenesis, Tumour stroma","lastPublishedDoi":"10.21203/rs.3.rs-8384416/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8384416/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e\u0026nbsp;The use of suitable radiotracers in positron emission tomography (PET) enables non-invasive quantification of tissue biomarkers in patients. Platelet-derived growth factor receptor beta (PDGFRβ) is a key mediator of fibrogenesis and tumour stroma biology, expressed on activated pericytes, fibroblasts, and cancer-associated stromal cells. These features make PDGFRβ an attractive target for imaging pathological tissue remodelling across fibrotic and malignant diseases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u0026nbsp;\u003c/strong\u003eHere, we report the development and clinical translation of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-DOTA-Cys-ATH001 ([\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001), a novel PET tracer targeting PDGFRβ. [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 was successfully produced using an automated in-house sequence on the ModularLab PharmTracer synthesis module. The process achieved high radiochemical purity (97 ± 0.4%), specific activity (2.4 ± 0.1 GBq/mg at the time of administration), reproducibility, and batch-to-batch consistency, ensuring suitability for clinical application. Translation into routine GMP production enabled a clinical trial in which [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 was administered to participants with liver metastases.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e\u0026nbsp;A robust, cGMP-compliant automated synthesis of [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 has been established, supporting its clinical translation as a novel PDGFRβ-targeting PET tracer for quantitative imaging of fibrogenesis. The ongoing IMAGINE-1 trial will evaluate PDGFRβ-PET for visualization of liver tumour stroma. Together, these applications position [\u003csup\u003e68\u003c/sup\u003eGa]Ga-ATH001 as a versatile tracer for assessing PDGFRβ-mediated pathophysiology across both fibrotic and oncologic indications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEU Trial registration:\u003c/strong\u003e\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eClinicalTrials.gov, NCT, NCT06562361, Registered 14 August 2024, Study Details | NCT06562361 | A Microdose Trial Investigating Binding of [68Ga]Ga-DOTA-CYS-ATH001 in Healthy Subjects and Different Patient Groups. | ClinicalTrials.gov\u003c/li\u003e\n \u003cli\u003eClinical Trials Information system: CTIS, EU CT 2025-522290-11-00, Registered 29 September 2025, IMAGINE 1 | 2025-522290-11-00 | CTIS.eu\u003c/li\u003e\n\u003c/ol\u003e","manuscriptTitle":"GMP-Compliant Manufacturing of [68Ga]Ga-ATH001 for a Human Microdose Trial Targeting Platelet-Derived Growth Factor Receptor Beta (PDGFRβ)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 01:03:11","doi":"10.21203/rs.3.rs-8384416/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":"4f9fa02e-fcc7-4a89-a307-8f171ce0a1e5","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-24T14:00:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 01:03:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8384416","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8384416","identity":"rs-8384416","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}