Automated Preparation of [68Ga]Ga-3BP-3940 on a Synthesis Module for PET Imaging of the Tumor Microenvironment

Summary

This research describes the automated process for [⁶⁸Ga]Ga-3BP-3940 production with the GAIA V2 synthesizer, for PET imaging of fibroblast activation protein. The results of quality control tests performed on three test batches are also presented.

Abstract

A fast, efficient method has been developed on the GAIA synthesis module for automated gallium-68 radiolabeling of 3BP-3940, a molecular imaging probe targeting the fibroblast activation protein for positron emission tomography imaging of the tumor microenvironment. The reaction conditions involved acetate buffer (final concentration: 0.1 M), methionine as an anti-radiolysis agent (final concentration: 5.4 mg/mL), and 30 µg of 3BP-3940, with heating for 8 min at 98 °C. A final purification step on a C₁₈ cartridge was necessary to obtain a radiolabeled product of high purity. In contrast, the generator-produced ⁶⁸Ga was used directly without a concentration step on a cation exchange cartridge. The production of three validation batches confirmed the method's reliability, allowing the synthesis of [⁶⁸Ga]Ga-3BP-3940 in 22.3 ± 0.6 min with high radiochemical purity (RCP), as determined by both radio-HPLC (99.1% ± 0.1%) and radio-TLC (99.2% ± 0.1%). The average radiochemical yield, based on RCP values measured by radio-HPLC, was 74.4% ± 3.3%. The stability of the radiolabeled product was demonstrated for up to 4 h after preparation. This protocol provides a reliable, rapid, and efficient methodology for the preparation of [⁶⁸Ga]Ga-3BP-3940, which can easily be transposed to a clinical setting.

Introduction

In recent years, targeting the tumor microenvironment (TME) has attracted considerable interest in diagnostic and therapeutic applications¹. The abundance of cell types, signaling molecules, and extracellular matrix (ECM) macromolecules within the TME offers a wide range of potential molecular targets². Among the resident and infiltrating host cells, cancer-associated fibroblasts (CAFs) form a distinct subset of fibroblasts within the TME, phenotypically different from normal fibroblasts. CAFs play crucial roles in tumor progression, metastasis, immune evasion, and therapy resistance through unique cellular and molecular....

Protocol

NOTE: This protocol involves working with radioisotopes. Anyone conducting this procedure must be properly trained in handling unsealed radioactive materials and must have approval from their institution's radiation safety officer. The automated synthesizer should be placed in a designated shielded hot cell. Any manual procedures involving radioactive materials should also be carried out in a shielded hot cell or behind appropriate radiation shielding.

1. Preparation of reagents

NOTE: The reagents required for the automated production of [⁶⁸Ga]Ga-3BP-3940 (see Table of Materials<....

Results

The synthesis process developed on the GAIA module allows fast ⁶⁸Ga radiolabeling of 3BP-3940 in 21-22 min. This protocol was designed to work with pharmaceutical grade ⁶⁸Ge/⁶⁸Ga generator GALLIAD, which produces 1.1 mL of ⁶⁸Ga eluate in 0.1 M HCl. The volume and molarity of the reaction buffer were finely tuned according to this amount of acid to obtain a reaction pH between 3.5 and 4, necessary for optimal radiolabeling⁴⁵. Thus, sodium acetate with a f.......

Discussion

This work presents a GMP-compliant automated preparation protocol for the synthesis of [⁶⁸Ga]Ga-3BP-3940 using a GAIA module and a GALLIAD generator. This method was adapted from protocols used in our center for gallium-68 radiolabeling of vectors such as PSMA ligands⁴⁴ and other FAP inhibitors^43,46 for clinical PET imaging, with slight modifications.

The production process was designed to be simple a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 µ filters	VWR	514-0515	For filtration of buffer and antioxidant solutions and final radiolabeling product
Acetonitrile for HPLC	Sigma Aldrich	34851-2.5L	For HPLC control of radiochemical purity
Ammonium acetate	Sigma Aldrich	238074	For the preparation of one of the mobile phases for TLC control
C18 column for HPLC	VWR	EQV-3C18-1503	For HPLC control of radiochemical purity
Calibrated dose calibrator (CRC25)	Capintec	-	For measuring the radioactivity of the final product and the various components of the module post-synthesis
Citrate buffer solution, pH 4	Thermofisher	258585000	Mobile phase for TLC controls
Eppendorf tube 5 mL Biopur	Sigma Aldrich	EP0030119479	For the preparation of buffer and antioxidant solutions
Extension line (30 cm)	Vygon	1159.03	For the connection of the generator to the tubing set
Gallium-68 generator	IRE Elit	-	For in situ generation of [68Ga]gallium chloride
Gamma counter (Hidex AMG)	Hidex	-	For half-life and radiochemical purity assessment
HPLC station	Shimadzu	-	For HPLC control of radiochemical purity
iTLC-SG plates	Agilent	SGI0001	For TLC control of radiochemical purity
L-methionine	AppliChem	A1340	For antioxidant solution preparation
Male/male adapter	Vygon	893.00	For the connection of the generator to the tubing set
Methanol	Sigma Aldrich	320390-1L	For the preparation of one of the mobile phases for TLC control
Needles (21G, Sterican)	B Braun	4657543B	For solution transfers prior to radiolabeling
pH paper	VWR	85409.600	To test the pH of the radiolabelling product
Pipette 1000 µL (Gilson PIPETMAN)	Fisher Scientific	12346132-1000	For precise liquid measurement and transfer
Pipette 200 µL (Gilson PIPETMAN)	Fisher Scientific	12326132-200	For precise liquid measurement and transfer
Pipette Tips, 100-1000 μL	Charles River	D1000IW	For precise liquid measurement and transfer
Pipette Tips, 2-200 μL	Charles River	D200IW	For precise liquid measurement and transfer
Radiochromatograph	Elysia-Raytest	-	For TLC control of radiochemical purity
Radiosensor for HPLC	Elysia-Raytest	-	For HPLC control of radiochemical purity
Reagents kit	ABX	RT-101	Provides ethanol 60%, NaCl 0.9%, WFI bag, C18 cartridge, 0.2 µ terminal filter, aeration needles, terminal needle and waste vial
Shielded container	LemerPax		For radiation attenuation of the radiolabeling product
Single-use plastic spatula	Corning	3005	For the preparation of reagents
Sodium acetate trihydrate EMPROVE	Sigma Aldrich	1.28204	For reaction buffer preparation
Sterile sealed vials (glass type 1)	Curium	TC-ELU-5	For final conditioning of buffer, antioxidant and radiolabeling solutions
Sterile tubing set	ABX	RT-01-H	For automated synthesis of [68Ga]Ga-3BP-3940
Sterile water for irrigation	B Braun	0082479E	For the preparation of one of the mobile phases for TLC control
Synthesis module (GAIA)	Elysia-Raytest	-	For automated synthesis of [68Ga]Ga-3BP-3940
Syringe (1 mL, low dead-volume)	B Braun	9166017V	For peptide in buffer conditionning and addition of methionine in NaCl 0.9%
Syringes (10 mL)	Becton Dickinson	309649	For methionine in NaCl 0.9% and conditionning
Syringes (3 mL)	Becton Dickinson	309658	For methionine and ethanol 60% conditionning
TLC migration tanks	Fisher Scientific	50-212-281	For TLC control of radiochemical purity
Trifluoroacetic acid (suitable for HPLC)	Sigma Aldrich	302031-100ML	For HPLC control of radiochemical purity
Tubes for gamma counter	-	-	For half-life and radiochemical purity assays preparation
Ultrasonic bath	Selecta	3000683	For sonication of prepared solutions
Vector molecule (3BP-3940)	MedChemExpress	HY-P10131	Vector molecule to be radiolabeled
Vial for HPLC with glass insert	Sigma Aldrich	29385-U and SU860066	For HPLC control of radiochemical purity
Vortex mixer	VWR	444-5900P	For stirring the prepared solutions
Water for HPLC	Sigma Aldrich	34877-2.5L-M	For HPLC control of radiochemical purity
Water for injection, 10 mL flasks	Aguettan	34009 370 641 0 1	For solutions preparation