Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jul 22:12:1628158.
doi: 10.3389/fmed.2025.1628158. eCollection 2025.

Specific reaction conditions for efficient automated 68Ga-radiolabeling of the FAP-2286 pseudopeptide on a GAIA® synthesizer

Maissa Ammour  1 , Jade Torchio  1 , Stéphane C Renaud  1 , Léa Rubira  1 , Cyril Fersing  1   2
Affiliations

Specific reaction conditions for efficient automated 68Ga-radiolabeling of the FAP-2286 pseudopeptide on a GAIA® synthesizer

Maissa Ammour et al. Front Med (Lausanne). .

Abstract

Introduction: Automated radiolabeling of gallium-68-labeled experimental radiopharmaceuticals is crucial for ensuring high reproducibility and regulatory compliance in clinical settings. FAP-2286, a promising DOTA-pseudopeptide targeting the tumor microenvironment, has demonstrated superior tumor retention compared to quinoline-based analogs, making it an attractive theranostic agent. This study aimed to optimize and automate the preparation of [68Ga]Ga-FAP-2286 on the GAIA® synthesizer, ensuring high radiochemical purity (RCP) and radiochemical yield (RCY).

Methods: Manual radiolabeling assays were initially performed to identify optimal reaction conditions, varying buffer, antioxidant, vector amount, heating time, and purification methods. The selected conditions were then adapted to an automated protocol using a GAIA® module. A strong cation exchange (SCX) cartridge for 68Ga pre-concentration and a solid-phase extraction (SPE) step for final purification were included in the process. RCY, RCP, and stability over 4 h were assessed using radio-HPLC and radio-TLC. Additionally, the applicability of the optimized automated method was evaluated for 3BP-3940, a structurally related pseudopeptide.

Results: Initial optimization studies identified sodium acetate buffer 0.1 M with methionine as an antioxidant, 25 μg of FAP-2286, and a 4-min heating time as the best manual radiolabeling conditions, achieving a RCP > 98%. In the automated synthesis, adjustments were made, including doubling the vector amount and extending heating to 9 min, resulting over three test-batches in a moderate RCY of 59.85 ± 3.73% and a RCP just over 94% up to 4 h after the end of synthesis. Importantly, the method was successfully transposed to [68Ga]Ga-3BP-3940, yielding better RCY (75.62 ± 11.76%), RCP and stability profiles (> 95.95% over 4 h).

Conclusion: This study established a robust, automated protocol for the synthesis of [68Ga]Ga-FAP-2286, ensuring high purity, reproducibility, and compatibility with clinical applications. The method's successful adaptation to 3BP-3940 highlights its versatility for such radiopharmaceuticals, supporting the broader implementation of automated theranostic agent production in nuclear medicine.

Keywords: FAP-2286; PET imaging; automated radiolabeling; gallium-68; radiopharmaceuticals; tumor microenvironment.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Chemical structure diagram of a complex organic compound. It includes various functional groups such as amides, esters, thiols, and an aromatic benzene ring. The variable X is shown as either CH2 for FAP-2286 or NH for 3BP-3940.
FIGURE 1
Chemical structure of the vector molecules FAP-2286 and 3BP-3940.
Diagram and photograph side by side. A: Schematic of a chemical synthesis apparatus with labeled components including sensors, filters, vials, a heating block, and a peristaltic pump. B: Photograph of the actual equipment setup, showing the Elysia-Raytest GAIA synthesis module with connected tubing and containers on a lab bench.
FIGURE 2
(A) Cassette-based scheme of the GAIA® synthesizer for [68Ga]Ga-FAP-2286. (B) Photograph of the set-up on the GAIA® module.
Flowchart of the synthesis process for Gallium-68 ([68Ga]) labeled FAP-2286. The left side lists 15 step-by-step procedures for the synthesis, starting with tubing set preparation and ending with final product vial release. The right side illustrates the setup, including equipment like the generator, SCX and SPE cartridges, reaction medium, and final product vial. Key stages include reaction medium preparation and washing processes, detailing specific conditions like temperatures and solution concentrations.
FIGURE 3
Flow chart for the automated [68Ga]Ga-FAP-2286 production process.
Bar graph showing RCP percentages determined by radio-HPLC for different buffer solutions. Sodium acetate 0.1 M (pH 4.2) has 90.89%, sodium acetate 0.5 M (pH 3.6) has 91.00%, sodium 1.5 M acetate (pH 3.9) has 73.39%, ammonium acetate 0.1 M (pH 4.5) has 92.73%, ammonium acetate 0.5 M (pH 3.6) has 77.76%, 1.5 M ammonium formate (pH 4) has 82.90%, sodium formate 0.5 M (pH 3.8) has 88.76%, sodium formate 1.5 M (pH 3.8) has 89.17%, HEPES 0.5 M (pH 3.5) has 92.56%, and HEPES 1.5 M (pH 4) has 86.70%.
FIGURE 4
Mean RCP values (determined by radio-HPLC) for [68Ga]Ga-FAP-2286 using buffers of different types and molarities.
Chart A is a bar graph showing the RCP determined by radio-HPLC for different anti-radiolysis compounds: no compound (90.89%), ascorbic acid (88.08%), gentisic acid (78.80%), and methionine (94.65%). Chart B is a line graph depicting the RCP over four hours for various sodium acetate solutions with additives: sodium acetate only decreases slightly, ascorbic acid shows a moderate decrease, gentisic acid shows a significant decline, and methionine remains relatively stable.
FIGURE 5
(A) Mean RCP values (determined by radio-HPLC) for [68Ga]Ga-FAP-2286 prepared in the presence of different antioxidant compounds. (B) Time course of mean RCP (determined by radio-HPLC) for [68Ga]Ga-FAP-2286 in the presence of different antioxidant compounds.
Bar chart showing the radiochemical purity (RCP) of FAP-2286 at different amounts: 12.5 micrograms at 93.51%, 25 micrograms at 98.12%, and 50 micrograms at 98.60%, determined by radio-HPLC. Error bars indicate variability.
FIGURE 6
Mean RCP values (determined by radio-HPLC) for [68Ga]Ga-FAP-2286 depending on the amount of vector molecule used for radiolabeling.
Bar chart showing the radiochemical purity (RCP) determined by radio-HPLC over different heating times. At 4 minutes, RCP is 98.63%; at 8 minutes, 98.12%; and at 12 minutes, 98.30%. Error margins are provided for each measurement.
FIGURE 7
Mean RCP values (determined by radio-HPLC) for [68Ga]Ga-FAP-2286 depending on the heating time of the reaction.
Bar chart comparing the mean percentage recovery and mean RCP for different types of SPE cartridges: C18, HLB, Strata-X, and CM. C18 shows 76.70% recovery and 98.09% RCP. HLB shows 75.05% recovery and 97.97% RCP. Strata-X shows 23.63% recovery and 93.94% RCP. CM shows 63.70% recovery and 94.92% RCP. Error bars indicate variability.
FIGURE 8
Mean RCP values (determined by radio-HPLC) and mean recovery (%) for [68Ga]Ga-FAP-2286 depending on the cartridge used for post-synthesis purification.
Three graphs labeled A, B, and C display data related to the detection of specific elements or compounds. Graph A features a red peak around 50 to 70 minutes, indicating signal intensity changes. Graph B shows a similar pattern with a green peak from 30 to 60 minutes. Graph C presents a singular, narrow blue peak at around 8 minutes, with cps values displayed. The x-axis represents time in minutes, and the y-axis indicates counts per second (cps) multiplied by one thousand.
FIGURE 9
Representative radio-TLC [(A) aqueous ammonium acetate 1 M in methanol (1:1); (B) aqueous sodium citrate 0.1 M pH 5] and radio-HPLC (C) spectra for [68Ga]Ga-FAP-2286 produced via automated method.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Schwenck J, Sonanini D, Cotton J, Rammensee H, La Fougère C, Zender L, et al. Advances in PET imaging of cancer. Nat Rev Cancer. (2023) 23:474–90. 10.1038/s41568-023-00576-4 - DOI - PubMed
    1. Adler S, Seidel J, Choyke P. Advances in preclinical PET. Semin Nucl Med. (2022) 52:382–402. 10.1053/j.semnuclmed.2022.02.002 - DOI - PMC - PubMed
    1. Ninatti G, Pini C, Lazar A, Gelardi F. The wings of progress: technological and radiopharmaceutical innovations in nuclear medicine. Eur J Nucl Med Mol Imaging. (2024) 51:3815–21. 10.1007/s00259-024-06913-5 - DOI - PubMed
    1. Kleynhans J, Ebenhan T, Sathekge M. Expanding role for gallium-68 PET imaging in oncology. Semin Nucl Med. (2024) 54:778–91. 10.1053/j.semnuclmed.2024.06.001 - DOI - PubMed
    1. Spang P, Herrmann C, Roesch F. Bifunctional gallium-68 chelators: past, present, and future. Semin Nucl Med. (2016) 46:373–94. 10.1053/j.semnuclmed.2016.04.003 - DOI - PubMed

LinkOut - more resources