A subscription to JoVE is required to view this content. Sign in or start your free trial.
This work describes the automated production of up to 1.7 GBq of [68Ga]Ga-FAPI-46 on the iPHASE MultiSyn synthesizer for PET imaging of fibroblast activation protein.
[68Ga]Ga-FAPI-46 is a promising new tracer for the imaging of fibroblast activation protein (FAP) by positron emission tomography (PET). Labeled FAP inhibitors (FAPIs) have demonstrated uptake in various types of cancers, including breast, lung, prostate, pancreatic and colorectal cancer. FAPI-PET also possesses a practical advantage over FDG-PET as fasting and resting are not required. [68Ga]Ga-FAPI-46 exhibits enhanced pharmacokinetic properties, improved tumor retention, and higher contrast images than the earlier presented [68Ga]Ga-FAPI-02 and [68Ga]Ga-FAPI-04. Although a manual synthesis protocol for [68Ga]Ga-FAPI-46 was initially described, in recent years, automated methods using different commercial synthesizers have been reported.
In this work, we describe the development of the automated synthesis of [68Ga]Ga-FAPI-46 using the iPHASE MultiSyn synthesizer for clinical applications. Initially, optimization of the reaction time and comparison of the performance of four different solid phase extraction (SPE) cartridges for final product purification were investigated. Then, the development and validation of the production of 0.6-1.7 GBq of [68Ga]Ga-FAPI-46 were conducted using these optimized parameters. The product was synthesized in 89.8 ± 4.8% decay corrected yield (n = 6) over 25 min. The final product met all recommended quality control specifications and was stable up to 3 h post synthesis.
Fibroblast activation protein (FAP) has become a prominent target for cancer imaging and therapy1,2. FAP is a specific marker of cancer-associated fibroblasts (CAFs), a stromal cell type constituting much of the microenvironment of solid cancers. CAFs play a key role in tumor growth, invasion, and metastasis3. They are found in most solid tumors, including breast, prostate, and pancreatic cancers1. By targeting FAP with small molecule inhibitors labeled with diagnostic or therapeutic radionuclides, selective non-invasive imaging and therapy of these cancers may be achieved4,5,6. FAP inhibitor (FAPI) molecules such as FAPI-02, FAPI-04, FAPI-46, and FAPI-74 labeled with 68Ga and 18F represent a class of quinoline-based FAP-targeting agents that were developed by teams at the Heidelberg University Hospital and the German Cancer Research Centre (DKFZ), Germany7,8,9,10,11, drawing on earlier work identifying the highly promising N-(4-quinolinoyl)-glycyl-(2-cyanopyrrolidine) scaffold for FAP inhibition12,13. More recently, cyclic-peptide FAP inhibitors such as FAP-228614 and 3BP-394015 have been developed for both imaging and therapy.
[68Ga]Ga-FAPI-04 has demonstrated uptake in 28 different types of cancers, including breast, lung, prostate, pancreatic, and colorectal cancer16. In addition to the practical advantage of not requiring fasting and resting9, FAPI-PET has shown a diagnostic advantage over (or a complementary role with) FDG-PET, such as in gastrointestinal, breast, ovarian, and liver cancer, in brain metastases of lung cancer17, and in cases where FDG findings are inconclusive18. 68Ga-labeled FAPI has also shown some interesting non-oncological applications in immune-related inflammatory diseases, such as fibrosis and rheumatoid arthritis19,20. The more recently presented [68Ga]Ga-FAPI-46, a variant of [68Ga]Ga-FAPI-04, which has a modified dodecane tetraacetic acid (DOTA) linkage, exhibits enhanced pharmacokinetic properties and improved tumor retention, resulting in higher-contrast images than those obtained using [68Ga]Ga-FAPI-02 or [68Ga]Ga-FAPI-046,10. The use of labeled FAP inhibitors for diagnosis and treatment is under international patent21 and the use of [68Ga]Ga-FAPI-46 is currently licensed22.
Reported preparations of [68Ga]Ga-FAPI-46 using either cyclotron-produced 68Ga or 68Ga eluted from a 68Ge/68Ga generator include the use of both manual protocols and automated synthesizers. A manual labeling protocol for [68Ga]Ga-FAPI-46 has been described, based on earlier reported methods for [68Ga]Ga-FAPI-02 and [68Ga]Ga-FAPI-0423,24. Although manual labeling does not require specialized equipment, this approach can lead to increased operator radiation dose and potential variability within production25; hence, the need to automate the production for routine clinical applications. Furthermore, the use of an automated synthesizer is more compliant with international GMP regulations. The preparation of [68Ga]Ga-FAPI-46 was developed on a variety of commercial synthesizers, using 68Ga from different generators26,27,28,29,30,31,32,33 or cyclotron-produced 68Ga34. The specifics of these automated methods are summarized in Supplemental Table S1 (Supplemental File 1). Automated synthesis methods for other 68Ga- and 177Lu-labeled FAP inhibitors have also been published in recent years35,36.
At the Sir Charles Gairdner Hospital RAPID Centre, we have developed and validated the preparation of [68Ga]Ga-FAPI-46 on the iPHASE MultiSyn synthesizer (hereafter referred to as the MS synthesizer). This synthesizer is used routinely in our laboratory, as well as at other production facilities in Australia for the preparation of 68Ga, 177Lu, and 89Zr-labeled radiopharmaceuticals37,38,39. The MS synthesizer is operated by downloading an Excel sequence step-list to its internal memory. This sequence step-list is user-friendly and easily modifiable. Furthermore, the synthesizer allows for mid-production interventions such as repeating steps, going back to previous steps, skipping steps, and pausing production when necessary. It is equipped with radiation detectors placed in strategic locations, allowing the user to monitor the production process in real time. The MS synthesizer is compatible with all commercial 68Ga generators and allows for single or double generator elution. The procedure described in this work utilizes 68Ga from one or two 68Ge/68Ga generator(s) and involves both prepurification of 68Ga and postpurification of the final product. Optimal reaction time and comparison of the performance of three different types of postpurification solid-phase extraction (SPE) cartridges to the one provided in the supplier's cassette, were also evaluated as part of this study.
CAUTION: This protocol involves the handling of radioactive materials. All personnel undertaking this work must be adequately trained in working with unsealed radioisotopes and have the approval of their institution's radiation safety officer. The automated synthesizer must be located in a dedicated shielded hot cell. Manual experiments must be performed in a shielded hot cell or behind radiation shielding. Preliminary experiments for the optimization of the reaction time and testing of various SPE cartridges are described in Supplemental Section 1 (Supplemental File 1).
1. Preparation of the MS synthesizer
2. Preparation of the reagents
NOTE: The reagents required for the automated production of [68Ga]Ga-FAPI-46 (see Table 1) were prepared in a clean room environment immediately prior to production.
3. Preparation of the synthesis cassette and cassette installation
4. Reagents, generator(s) line(s), and final product vial Installation (see Figure 1A,C and Figure 1B,C if using dual generator elution)
5. Synthesizer preliminary steps prior to radiolabeling
6. Automated radiolabeling to produce [68Ga]Ga-FAPI-46
NOTE: The automated synthesis is initiated by performing step 5.2. Figure 1D describes the radiolabeling reaction to produce [68Ga]Ga-FAPI-46. A representative screenshot of the synthesizer's interface and a typical radioactivity profile are shown in Figure 1A,B and Figure 2, respectively. For production using dual generator elution, the synthesizer will prompt the user to remove the empty Syringe B after elution of the first generator and install another syringe containing 5 mL HCl 0.1 M on the syringe driver for the elution of the second generator.
Table 1: Reagent preparation for the production of [68Ga]Ga-FAPI-46. Please click here to download this Table.
Figure 1: Schematic of the synthesizer user interface; cassette and reagent setup for automated radiosynthesis of [68Ga]Ga-FAPI-46. (A) Single generator production setup. (B) Dual generator production setup. (C) Reagent positions for automated production of [68Ga]Ga-FAPI-46 using the MS radiosynthesizer. (D) [68Ga]Ga-FAPI-46 radiolabeling scheme. Abbreviations: FAPI = fibroblast activation protein inhibitor; Mn = manifold n; Wn = waste outlet n; Vn = valve n; R = reactor vacuum; Gn = gas inlet n. Please click here to view a larger version of this figure.
Figure 2: Synthesizer typical radioactivity profile for the automated synthesis of [68Ga]Ga-FAPI-46. Abbreviations: FAPI = fibroblast activation protein inhibitor; SPE = solid phase extraction. Please click here to view a larger version of this figure.
7. Dispensing [68Ga]Ga-FAPI-46 for quality control and shipment
8. Quality control of [68Ga]Ga-FAPI-46
NOTE: The quality control tests described below were performed in accordance with procedures described in the European Pharmacopoeia40.
9. Stability testing
The radiolabeling efficiency assessed between 5 and 20 min of reaction at 95 °C is reported in Table 2. The postpurification SPE cartridges HLB 30 mg, Strata X 60 mg, and Sep-Pak C18 Plus Short 360 mg showed very similar recoveries (94.3 ± 0.5% decay-corrected [DC]) (Table 3). In our hands, the recovery off the HLB 225 mg was much lower (63.8 ± 3.5% DC).
Table 2: Summary of the radiochemical conversion of the crude reaction measured by T...
This work describes the reliable and high-yielding production of [68Ga]Ga-FAPI-46 on the MS synthesizer for clinical applications. The preliminary workup of this protocol tested, in the same series of experiments, different reaction times for the synthesis of [68Ga]Ga-FAPI-46 as well as four different SPE cartridges for the purification of the final product. In order to (i) reduce the radiation dose to the operator and (ii) re-create the conditions in which the routine production of [68Ga...
The authors have no commercial partnerships or funding sources that would result in a real or perceived conflict of interest relating to this work to disclose.
The authors acknowledge and thank SOFIE Biosciences Inc. for supplying the FAPI-46 chemical precursor and [natGa]Ga-FAPI-46 standard, the Charlies Foundation for Research for financial support, Stan Poniger from iPHASE Pty Ltd, and the Radiopharmaceuticals Production team (RAPID) at the Medical Technology and Physics Department at Sir Charles Gairdner Hospital for their scientific and technical support. The authors also acknowledge the assistance of the WA National Imaging Facility Node, which is supported by infrastructure funding from the Western Australian State Government in partnership with the Australian Federal Government, through the National Collaborative Research Infrastructure Strategy (NCRIS) capability.
Name | Company | Catalog Number | Comments |
0.1 M Hydrochloric acid (HCl) ultra pure | ABX advanced biochemical compound (Radberg, Germany) | HCl-103-G | Used for generator(s) elution |
Ammonium acetate | Sigma Aldrich Pty Ltd (NSW, Australia) | A1542-250G | Used to make iTLC mobile phase |
C18 SepPak Plus short (360 mg) | Waters | WAT020515 | Post-purification silica SPE |
Chromolith Performance RP-18 endcapped 100-4.6 monolithic | Merck Pty Ltd (Victoria (Australia) | 1021290001 | HPLC RP-18 endcapped column, used for HPLC quality control |
Dose calibrator | Capintec | CRC-15PET | Used to calibrate and measure 68Ga activity |
Dual scan-RAM | LabLogic Limited (VA, USA) | SR-1A | Radio-TLC scanner to analysise the iTLC paper |
FAPI-46 precursor (GMP) | ABX advanced biochemical compound (Radberg, Germany) | 3601.0000.050 | Peptide precursor |
Fill ease Sterile vacuum vial (10 mL) | HUAYI iosotopes | SVV-10C | Used for sterility and retention samples |
Fill ease Sterile vacuum vial (25 mL) | HUAYI iosotopes | SVV-25A | Used for final product |
Ga68 peptide radiolabelling with generator pre-purification | iPHASE Technologies (Melbourne, Australia) | MSR-120G-(RK-3296) | Reagent set |
Ga68 radiolabeling with generator prepurification | iPHASE Technologies (Melbourne, Australia) | MSH-120 | Hardware Cassette + ancillaries set |
Gas chromatography (GC) system | Agilent technologies (Vic, Australia) | G2630A | Used to measure residual solvent |
GS Standard source (Ba133) | Global Medical Solutions Pty Ltd (Australia) | D-102-19 | Used to calibrate the Gamma Spectometer |
GS Standard source (Co60) | Global Medical Solutions Pty Ltd (Australia) | 1559-84 | Used to calibrate the Gamma Spectometer |
High performance liquid chromatography (HPLC) system | Shimadzu Scientific Instruments (NSW, Australia) | LC-20 | HPLC equipment |
Hydrophobic air vent needle | Baldwin Medical (Victoria, australia) | 1088 | Used with final product vial |
IGG100 | Eckert & Ziegler Isotope Products (Berlin, Germany) | IGG100-65M-NT | 68Ge/68Ga generator |
5 mL syringe (Injekt luer lock solo syringe) | B Braun (Melsungen, Germany) | 4606710V | Polypropylene (PP)/polyethylene (PE) syringes, free of latex, PVC, and silicone oil free syringe used for reagents |
iTLC-SG paper | Agilent technologies (Vic, Australia) | SGI0001 | Used to for iTLC analysis |
LabLogic software (LAURA) | LabLogic Limited (VA, USA) | LAURA software version 6.1 | Used to for radio-TLC analysis |
L-Ascorbic acid Trace select | Fluka Sigma | 05878-100G | Used as a radical scavenger in the reaction mixture |
Lichrosolv Acetonitrile (ACN) | Sigma Aldrich Pty Ltd (NSW, Australia) | 1.00030.2500 | Used to make HPLC organic mobile phase |
Lichrosolv Water | Sigma Aldrich Pty Ltd (NSW, Australia) | 1.15333.2500 | Used to make HPLC aqueous mobile phase |
Methanol (MeOH) | Sigma Aldrich Pty Ltd (NSW, Australia) | 1060182500 | Used to make iTLC mobile phase |
Na+I- detector | LabLogic Limited (VA, USA) | 1"NaI / PMT | Radiodetector used for radio-HPLC |
Oasis HLB (30 mg) | Waters (Milford, MA, USA) | WAT094225 | Postpurification copolymer SPE |
Oasis HLB Plus short (225 mg) | Waters (Milford, MA, USA) | 186000132 | Postpurification copolymer SPE |
pH strips | Thermo Fisher Scientific Australia Tty Ltd | 90424 | Used to measure product pH |
PS detector | LabLogic Limited (VA, USA) | PS plastic/PMT | Radiodetector used for radio-TLC |
Safe Lock tube (1.5 mL) | Eppendorf | 0030 120.086 | Used for quality control samples |
(+)-Sodium L-ascorbate | Merck Pty Ltd (Victoria (Australia) | 11140-250G | Stabilizer of the final product |
Sodium chloride (NaCl) solution (saline) | Pfizer | PS111 | 0.9%, for injection, USP grade |
Sterican 100 Needles | B Braun (Melsungen, Germany) | 4667093 | Used for final product |
Sterile syringe filter (0.22 µm) | Millipore Sigma (Burlington, MA, USA) | SLGSV255F | Millex-GV |
Strata SCX (in Hardware cassette kit) | Phenomenex inside hardware kit from iPHASE Technologies (Melbourne, Australia) | MSH-120 | Prepurification silica SPE inside Hardware Cassette |
Strata X (in Hardware cassette kit) | Phenomenex inside hardware kit from iPHASE Technologies (Melbourne, Australia) | MSH-120 | Postpurification silica SPE inside Hardware Cassette |
Trace Select Water for trace analysis | Honeywell Riedel-de-Haen | 95305-2.5L | Used for reaction mixture and to precondition the prepurification SPE cartridge |
Trifluoracetic acid (TFA) | Sigma Aldrich Pty Ltd (NSW, Australia) | 302031-10X1mL | Used to make HPLC aqueous mobile phase |
Ultra Fine insulin syringe (0.5 mL) | BD | 326769 | Used for dispensing quality control samples |
Vented filter Cathivex-GV 0.22 µm, low protein binding Durapore PVDF membrane | Merk Millipore (Cork, Ireland) | SLGV02505 | Used to filter the final product |
Request permission to reuse the text or figures of this JoVE article
Request PermissionThis article has been published
Video Coming Soon
Copyright © 2025 MyJoVE Corporation. All rights reserved