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

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.

FIGURE 1

Chemical structure of the vector molecules FAP-2286 and 3BP-3940.

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.

FIGURE 3

Flow chart for the automated [⁶⁸Ga]Ga-FAP-2286 production process.

FIGURE 4

Mean RCP values (determined by radio-HPLC) for [⁶⁸Ga]Ga-FAP-2286 using buffers of different types and molarities.

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.

FIGURE 6

Mean RCP values (determined by radio-HPLC) for [⁶⁸Ga]Ga-FAP-2286 depending on the amount of vector molecule used for radiolabeling.

FIGURE 7

Mean RCP values (determined by radio-HPLC) for [⁶⁸Ga]Ga-FAP-2286 depending on the heating time of the reaction.

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.

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.