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

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.

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.

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 characteristics³. These mesenchymal cells exhibit an activated phenotype marked by the expression of fibroblast activation protein (FAP). Molecularly, CAFs secrete a complex array of cytokines, chemokines, growth factors (e.g., TGF-β, IL-6, and CXCL12), and ECM proteins (e.g., collagen, fibronectin), which remodel the ECM and foster a pro-tumorigenic environment⁴.

As a highly specific protein that is overexpressed and localized on the extracellular surface of the CAF membrane, FAP displays all the characteristics of a reliable molecular target, especially for nuclear medicine and radiopharmaceutical applications⁵. In this context, quinoline-based small molecule inhibitors of FAP (FAPI), functionalized with a DOTA group, were developed and quickly introduced into clinical use^6,7,8. Specifically, FAPI-04 and FAPI-46 radiolabeled with gallium-68 (β⁺ emitter, t_1/2 = 68 min) for positron emission tomography (PET) imaging have demonstrated significant value in fibrotic diseases, cardiology, and oncology^8,9, particularly for cancers where [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) has limited utility¹⁰. However, while their contributions to oncology and nonmalignant diseases imaging are undeniable, small molecule FAPIs exhibit certain limitations for targeted radionuclide therapy (TRT) applications, particularly due to their suboptimal intratumoral residence time, which can lead to unintended irradiation of healthy tissue¹¹. To address this issue, several strategies have been explored, such as the design of multivalent ligands^11,12 or the use of therapeutic radionuclides with short half-lives^13,14,15. New molecular scaffolds with a high affinity for FAP and triggering a high proportion of cell internalization have also been developed.

One of these is the pseudopeptide derivative FAP-2286. It contains a 7-amino acid sequence, cyclized and linked to a DOTA chelator by a 1,3,5-benzenetrimethanethiol moiety¹⁶. An initial study in humans demonstrated that [⁶⁸Ga]Ga-FAP-2286 exhibits a biodistribution profile similar to [⁶⁸Ga]Ga-FAPI-46, with slightly higher physiological uptake in the liver, kidneys, and heart¹⁷. In this study, 64 patients, primarily with cancers of the neck, liver, stomach, pancreas, ovaries, and esophagus, underwent PET imaging with [⁶⁸Ga]Ga-FAP-2286 for cancer staging or detection of recurrence: uptake of [⁶⁸Ga]Ga-FAP-2286 was notably higher than [¹⁸F]FDG in primary tumors, lymph node metastases, and distant metastases, enhancing image contrast and lesion detectability. All primary tumors were visible with [⁶⁸Ga]Ga-FAP-2286 PET/CT, whereas [¹⁸F]FDG PET/CT missed almost 20% of the lesions. For involved lymph nodes, detection rates were higher with [⁶⁸Ga]Ga-FAP-2286, as well as for bone and visceral metastases. Another study in a smaller group of 21 patients with a variety of cancer diseases also demonstrated the excellent sensitivity of this imaging agent, reflecting the diagnostic efficiency of [⁶⁸Ga]Ga-FAP-2286¹⁸. More specific studies have focused on a single type of cancer, such as urothelial or lung cancer, highlighting once again the high potential of [⁶⁸Ga]Ga-FAP-2286 for clinical molecular imaging^4,5. Regarding therapy, a preliminary study investigated the use of FAP-2286 radiolabeled with lutetium-177 (β^- emitter, t_1/2 = 6.7 d) in 11 patients with diverse progressive, metastatic cancers¹⁹. Most patients received two treatment cycles spaced 8 weeks apart, and the average administered dose per cycle was 5.8 ± 2.0 GBq of [¹⁷⁷Lu]Lu-FAP-2286. The drug demonstrated prolonged intratumoral retention, with an effective half-life of approximately 44 h in bone metastases. Given the acceptable side effects, these findings paved the way for larger-scale clinical trials: the safety and efficacy of [¹⁷⁷Lu]Lu-FAP-2286 are currently being assessed in the phase 1/2 LuMIERE clinical trial, sponsored by Novartis (NCT04939610)^7,8. Further smaller-scale research protocols are documented in the literature^9,20, and multiple case reports have been published^{21,22,23,24,25,26}, demonstrating the efficacy and excellent tolerability of this TRT.

Minimal structure modifications made on FAP-2286 led to the optimized analog 3BP-3940 (Figure 1)²⁷. Although scientific literature on this vector molecule remains limited, early studies have been conducted for both imaging and therapeutic applications. A preliminary report describes the use of [⁶⁸Ga]Ga-3BP-3940 in 18 patients with various end-stage metastatic carcinomas and concludes that this radiopharmaceutical is a suitable PET imaging agent, emphasizing its excellent tumor-to-background ratio and very low kidney uptake²⁸. In another work, a single pancreatic cancer patient with liver metastases received 150 MBq of [⁶⁸Ga]Ga-3BP-3940 for PET imaging, which demonstrated intense uptake in the primary tumor and metastatic lesions²⁹. The same patient subsequently received a single dose of 9.7 GBq of [¹⁷⁷Lu]Lu-3BP-3940 for TRT. The treatment was well tolerated, with no significant changes in vital signs or biological parameters. A different study presented the initial human results of a theranostic approach using 3BP-3940: patients were selected with [⁶⁸Ga]Ga-3BP-3940 PET imaging and then received 3BP-3940 labeled with different isotopes (¹⁷⁷Lu, ⁹⁰Y, or ²²⁵Ac), administered alone or in tandem combinations (e.g., ¹⁷⁷Lu + ²²⁵Ac) in 1-5 treatment cycles³⁰. Outcomes included one complete remission, four partial remissions, three stable diseases, and 12 disease progressions. The cohort's (n = 28) median overall survival was 9 months from the start of TRT.

Figure 1: Chemical structure of [⁶⁸Ga]Ga-3BP-3940. Please click here to view a larger version of this figure.

The ⁶⁸Ga radiolabeling process for experimental radiopharmaceuticals such as FAP-2286 and 3BP-3940 generally involves a synthesis module to automate the preparation step. Notably, method automation ensures process robustness and GMP compliance and minimizes operator radiation exposure in comparison with manual preparation methods^31,32,33. In many cases, such a protocol is expected by regulatory authorities as a part of an investigational medicinal product dossier (IMPD) before authorizing a center to manufacture the corresponding experimental radiopharmaceutical³⁴. To date, very little detailed information on the automated ⁶⁸Ga radiolabeling of anti-FAP pseudopeptides is available in the literature^{29,35,36,37,38}. Moreover, the data reported generally applies only to a given model of synthesizer. The type of ⁶⁸Ga generator used can also bring certain specificities, as the different commercially available solutions are characterized by specific volumes of ⁶⁸Ga³⁺ eluate in HCl (usually 0.1 M), which can have a direct impact on automated radiolabeling conditions.

In this context, we present a detailed protocol for the rapid and efficient automated radiolabeling of the pseudopeptide 3BP-3940 with ⁶⁸Ga, using the GAIA V2 synthesis module. This synthesizer relies on the use of a tubing set comprising three ramps of five manifolds each, connected to a peristaltic pump to control fluid flow. It also features a vial oven for reaction medium heating, several radioactivity probes, and a pressure sensor to monitor these parameters within the system. Although not as widespread as some other models, this automaton is used routinely in our center and is installed in a growing number of facilities^{31,39,40,41,42,43,44} . A GALLIAD ⁶⁸Ge/⁶⁸Ga generator was used in this work without prepurification of the ⁶⁸Ga eluate. This method is designed to offer a robust, fast, and convenient solution for the production of [⁶⁸Ga]Ga-3BP-3940, also optimizing radiation protection for operators during radiolabeling. This is also the first preparation protocol for this radiopharmaceutical to be reported on this specific synthesizer model, and in such detail.

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) were prepared in a radiopharmaceuticals preparation unit (GMP grade C clean room). Reagents can be prepared in any order and up to 2 h prior to synthesis.

1. Preparation of buffer solution (sodium acetate 0.8 M)
   1. Acquire the following raw material: sodium acetate trihydrate EMPROVE API Ph Eur, BP, JP, USP, FCC, E262.
   2. In a sterile container (e.g., microcentrifuge tube, 5 mL) correctly identified, weigh an exact mass of sodium acetate trihydrate close to 544.32 mg using a precision balance.
   3. Using a calibrated micropipette and sterile cones, solubilize sodium acetate in a volume of water for injection (WFI) close to 5 mL so that WFI is as described below.
      ​WFI volume = (weighed buffer mass x 5)/544.32
   4. Vortex the solution and run it through an ultrasonic bath (40 kHz, ~1 min) to facilitate solubilization.
   5. Using a 10 mL syringe equipped with a 21G needle, withdraw the buffer solution. Install a 0.22 µm filter between the syringe and a new needle, then filter the buffer solution directly into a properly identified sterile sealed vial (e.g., TC-ELU 5) after disinfection of its septum.
2. Preparation of anti-radiolysis compound solution (methionine 10 mg/mL)
   1. Acquire the following raw material: L-Methionine (Ph. Eur., USP) pure, pharma grade.
   2. In a sterile container (e.g., microcentrifuge tube 5 mL) correctly identified, weigh an exact mass of L-methionine close to 50 mg using a precision balance.
   3. Using a calibrated micropipette and sterile cones, solubilize L-methionine in a volume of WFI close to 5 mL so that WFI is as described below.
      WFI volume = (weighed buffer mass x 5)/50
   4. Vortex the solution and run it through an ultrasonic bath (40 kHz, ~1 min) to facilitate solubilization.
   5. Using a 10 mL syringe equipped with a 21G needle, withdraw the methionine solution. Install a 0.22 µm filter between the syringe and new needle, then filter the solution directly into a properly identified sterile sealed vial (e.g., TC-ELU 5, Curium) after disinfection of its septum.

2. Preparation of equipment for quality controls

1. pH control of the final product
   1. Behind an appropriate protective shield, position a strip of pH paper for subsequent checking of the final product.
2. Radiochemical purity control by radio-TLC
   1. Prepare two TLC migration tanks containing appropriate mobile phases. Mobile phase A: citrate buffer 0.1 M pH 4 in water, with expected Rf = 0-0.2 for [⁶⁸Ga]Ga-3BP-3940 and Rf = 0.8-1 for free ⁶⁸Ga³⁺. Mobile phase B: ammonium acetate buffer 1 M in a 1:1 mixture of water and methanol (v/v), with expected Rf = 0-0.2 for [⁶⁸Ga]gallium colloids and Rf = 0.8-1 for [⁶⁸Ga]Ga-3BP-3940.
   2. Behind an appropriate protective shield, position the two TLC migration tanks next to two iTLC-SG plates for subsequent control of the RCP of the final product.
   3. Switch on the radiochromatograph and open the associated acquisition software on the operating computer. Pre-enter the identification information for the first analysis to be performed.
3. Radiochemical purity control by radio-HPLC
   1. Check that the column model required for the analysis (C₁₈ ACE Equivalence) is installed on the system.
   2. Prepare fresh mobile phase for HPLC, i.e., water for HPLC + 0.1% TFA (solvent A) and acetonitrile + 0.1% TFA (solvent B). Connect the solvent A and solvent B bottles to the corresponding lines on the radio-HPLC.
   3. Turn on the operating computer and connect to the control software.
   4. If necessary, open the purge valve on the pump module and purge the lines that will be used for analysis (i.e., line A and line B). Close the purge valve after this operation.
   5. Allow the system to equilibrate with a proportion of solvent equal to the start of the gradient (i.e., 95% A/5% B) with a 0.6 mL/min flow for at least 20 min. For the [⁶⁸Ga]Ga-3BP-3940 HPLC analysis sequence, use the flow rate maintained at 0.6 mL/min, and program the mobile phase gradient from 0.1% TFA in water (A) to 0.1% TFA in acetonitrile (B) as follows: 0 - 1 min 95/5 A/B; 1 - 8 min linear gradient from 95/5 A/B to 60/40 A/B; 8 - 9 min 60/40 A/B; 9 - 10 min linear gradient from 60/40 A/B to 95/5 A/B; 10 - 12 min 95/5 A/B.
   6. Select the analytical method with the mobile phase gradient described in step 2.3.5., then pre-enter the identification information for the first analysis to be performed.
   7. In a shielded container, prepare an HPLC vial with a glass insert to receive the sample to be analyzed and position it behind an appropriate protective shield.

3. Preparation of the synthesis module

1. Turn on the power and light of the shielded cell. Turn on the laptop controlling the synthesizer and log on to the module software.
2. If required, remove the old kit from the synthesizer and dispose of waste in the appropriate containers.
3. Perform cleaning of the inside of the shielded cabinet housing the synthesis module and of the synthesizer itself in accordance with applicable hygiene standards.
4. On the synthesizer software, press User, then select a username and enter the associated password in the pop-up window. Press Method and select the automated protocol previously configured for the [⁶⁸Ga]Ga-3BP-3940 synthesis.
5. Press Preparation and input the synthesis title, vector batch number, reagent kit batch number and any comments in the appropriate text box. A checklist with the various kit preparation steps can also be set up and followed at this stage (Supplementary Figure 1).

4. Preparation of the synthesis cassette and cassette installation

1. Clean the radiopharmacy lab workbench with a wipe and the appropriate detergent disinfectant and place a sterile drape over the workbench.
2. Obtain a sterile [⁶⁸Ga]Ga labeling cassette (reference RT-01-H) and a reagents kit (Supplementary Figure 2). The 3 ramps of the tubing set are identified as A, B, and C from left to right on the module; the manifolds of each ramp are numbered from 1 to 5 from top to bottom (ramps A and C) or from left to right (ramp B).
3. Assemble the cassette following the steps below.
   1. Unwrap the [⁶⁸Ga]Ga cassette envelope, check for any damage, and tighten each Luer connection on the cassette. Remove all the spike caps.
   2. Using a 5 mL Luer Lock syringe and 21G needle, draw up 5 mL of ethanol absolute from the reagent kit and pass it very slowly over the C₁₈ cartridge, then draw up 5 mL of WFI (not supplied in the kit) and pass it very slowly over the same C₁₈ cartridge to precondition it.
   3. Position ramp A of the tubing set on the synthesis module and turn the two latches to hold the ramp in place. Connect the free end of the vertical A1 tubing to a 19G needle and insert it into the waste vial.
   4. Add a venting needle to the waste vial and position it backward, optimally behind the shielded container that will receive the evacuation vial. Place a 0.22 µm filter in position A4.
   5. Connect the horizontal A1 tubing to the pressure sensor at the bottom left of the module front panel.
   6. Using a male/male adaptor, connect in horizontal A5 position a 30 cm extension ended by a 0.22 µ terminal filter and an 80 mm 20G needle.
   7. Insert the above-mentioned 20G needle into a sealed, sterile evacuation vial (e.g., TC-ELU 5), add an aeration needle and position the evacuation vial in its shielded container.
   8. Place the tubing line connecting vertical A1 to vertical C1 behind the retaining hooks above ramp B.
   9. Connect horizontal manifolds A2 and B1 with a short extension line (either not included in the kit, or the one initially connected in position C5 horizontal), using the adapter already mounted in position A2.
   10. Position ramp B on the synthesis module and turn the two latches to hold the ramp in place.
   11. Connect the pre-conditioned C₁₈ cartridge to the horizontal C2 position, keeping the adapter connecting the horizontal B5 valve to C2 on its left.
   12. Position ramp C on the synthesis module and turn the two latches to hold the ramp in place.
   13. Using a male/male adapter, connect a 50 cm extension line from horizontal C5 to the GALLIAD ⁶⁸Ga generator.
   14. Place the stained-glass reaction vial of the tubing set in the oven of the module. Carefully place the tubing from vertical A5 to vertical C5 in the peristaltic pump, close the pump, checking that the tubing is correctly positioned, and pass the tubing through the activity sensor on the left-hand side of the pump.
4. Perform an intermediate check to ensure that the cassette and tubing are assembled on the radiosynthesizer as shown in Figure 2, without reagents yet.

Figure 2: Synthesis module configuration. (A) Setup for automated synthesis of [⁶⁸Ga]Ga-3BP-3940 on the synthesis module. (B) Details on the reagent positions for automated production of [⁶⁸Ga]Ga-3BP-3940 using a GAIA synthesis module. Please click here to view a larger version of this figure.

5. Reagents installation

1. Once the ramps have been installed on the module, install the reagents as described below.
   1. Connect the WFI 250 mL bag from the reagent kit to the C4 tubing using the Spike adaptor, and hang the bag on the dedicated hook on the right-hand side of the module.
   2. Using a 3 mL three-piece Luer Lock syringe equipped with a 20G needle, withdraw 1.5 mL of the previously prepared L-methionine 10 mg/mL solution, connect the syringe to the horizontal C1 tubing and hang the syringe in the dedicated slot on the right-hand side of the module. Leave approximately 2 mL of air between the liquid surface and the syringe plunger seal to ensure complete liquid transfer.
   3. Using a 1 mL syringe with a 20G needle, withdraw 750 µL of the previously prepared 10 mg/mL L-methionine solution and inject into the 0.9% NaCl vial (blue crimp) from the reagent kit, after disinfection of the septum.
   4. Using a 10 mL three-piece Luer Lock syringe with a 20G needle, withdraw the contents of the 0.9% NaCl + L-methionine vial (blue crimp), adjust to 8.6 mL, remove the Spike in position B4, then connect the syringe in B4 instead. Leave approximately 2 mL of air between the liquid surface and the syringe plunger seal to ensure complete liquid transfer.
   5. Using a 3 mL three-piece Luer Lock syringe with a 20G needle, withdraw the contents of the 60% ethanol vial (orange crimp) after disinfection of the septum. Check that the volume is at least equal to 1.5 mL, remove the Spike in position B5, then connect the syringe to B5 instead. Leave approximately 2 mL of air between the liquid surface and the syringe plunger seal to ensure complete liquid transfer.
   6. Using a low dead volume 1 mL syringe with a 20G needle, withdraw 0.25 mL of the 0.8 M sodium acetate buffer solution previously prepared and inject into the vial containing 30 µg of 3BP-3940 to solubilize it by successive injections/re-aspirations cycles. Withdraw the 0.25 mL solution into the same syringe, disconnect the needle, and place the buffer + vector syringe in B3. Leave approximately 0.25 mL of air between the liquid surface and the syringe plunger to ensure complete liquid transfer.
2. Perform a final check before the start of the synthesis as described below.
   1. Verify that all the reagents are connected to the cassette or tubing, as shown in Figure 3.
   2. Ensure that each of the kit's connections is sufficiently tight. Make sure no tubing is pinched or angled.
3. The system is ready. Close the front panel and switch on the ventilation of the shielded cell.

Figure 3: Kit setup. Final installation of the tubing set and reagents on the synthesizer for the radiolabeling of 3BP-3940 with ⁶⁸Ga. Please click here to view a larger version of this figure.

6. Automated radiolabeling sequence for [⁶⁸Ga]Ga-3BP-3940 production

1. Click Run Synthesis when all reagents are placed on the ramps and all information is correctly recorded in the software.
2. A typical profile of radioactivity distribution in the tubing set throughout the synthesis sequence is shown in Figure 4. Follow the successive steps in the automated method for preparing [⁶⁸Ga]Ga-3BP-3940 as described below.
   1. Kit integrity tests: after a brief purge of the tubing towards the waste vial (essentially to remove any remaining liquid in the C₁₈ cartridge), the system increases its internal pressure by opening A4 and pumping filtered air via the peristaltic pump. As soon as the pressure sensor connected to A1 detects a tubing pressure > 1500 mbar, the system closes, and the peristaltic pump stops. If the pressure drop does not exceed 400 mbar over 15 s, assess the integrity test of the kit as successful and begin synthesis. If the test fails, suspect a system leak and abort the synthesis sequence.
   2. Addition of vector to reaction vial: Ensure that the 3BP-3940 solubilized in the buffer solution is drawn into the reaction vial in around 30 s, through manifold B2.
   3. Addition of L-methionine to reaction vial: Ensure that the methionine solution contained in the syringe at C1 is transferred to the reaction vial via manifold B2, down ramp C and then up ramp A to branch off at ramp B by manifold A2.
   4. Activation of C₁₈ cartridge with WFI: Ensure that the WFI contained in the bag at C4 is passed over the C₁₈ cartridge, also rinsing ramps A, B, and C on the paths followed by the vector and methionine solutions. The same routes are then flushed with filtered air to ensure that no water remains in the tubing for the subsequent steps.
   5. Generator elution in reaction vial (manual intervention): To elute the GALLIAD generator, turn the green knob on top of the generator to 90° to the loading position before waiting for 10-20 s: this time allows the generator to prepare a fixed eluate volume of 1.1 mL. Return the green knob to its initial position, and click the prompter on the Control software. The peristaltic pump then draws the ⁶⁸Ga eluate into the reaction vial over a 3 min period. Over this time interval, the temperature of the reaction vial is gradually raised to 60 °C over 1 min, then to 90 °C over 2 min, to reach the set temperature for the radiolabeling step more quickly.
   6. Radiolabeling: Check that at the start of the radiolabeling step, a setpoint temperature of 120 °C is applied for 30 s to enable rapid, efficient heating. Check that the setpoint temperature is then adjusted to 98 °C for 2 min. Due to the heating and pressure rise in the reaction vial, some of the reaction medium tends to flow back up into the tubing connected to B2. To limit this, a 10-second line purge is set after 2 min heating; then radiolabeling continues for around 5.5 min.
   7. Trapping on C₁₈ cartridge: Ensure that filtered air is pumped into the reaction vessel by C3. The pressure exerted on the surface of the liquid allows the reaction medium to flow upwards through the tubing connected at B2 and then to the SPE cartridge. The reaction vial is then rinsed with WFI from the bag at C4, and this rinsing liquid is transferred to the SPE cartridge in the same way. The cartridge is eventually rinsed with fresh WFI and flushed with filtered air.
   8. Elution of C₁₈ cartridge and formulation (addition of NaCl 0.9% + L-methionine): The SPE cartridge initially retains the radiolabeled product and any gallium colloids and allows free ⁶⁸Ga³⁺ to pass through. To elute the cartridge and recover [⁶⁸Ga]Ga-3BP-3940 in the terminal vial, ensure successive fractions of ethanol 60% and methionine ~0.9 mg/mL in NaCl 0.9% (3x 0.5 mL each) are drawn over the cartridge for elution. The entire contents of the syringe at B4 are then passed through the C18 cartridge in a formulation step to achieve a final ethanol concentration of <10% in the terminal vial.
   9. Filter integrity test (manual intervention): After synthesis and removal of the terminal vial, follow the prompt that asks to connect the terminal filter to the waste vial and remove the venting filter from the waste vial in order to check the integrity of the terminal filter using a bubble point test. During the bubble point test, the filter is initially wetted by WFI for 30 s. After purging the lines, pressure in the system is slowly increased up to >2500 mbar over <2 min, during which the synthesizer checks that there is no pressure increase on the outlet of the filter. Then, the system slowly increases the pressure to check the bubble point. The synthesizer will finally record the pressure when pressure increases on the filter outlet to determine the bubble point pressure.
3. At the end of the synthesis, the automaton software generates a synthesis report describing the course of the radiolabeling and tracking the temperatures in the reaction vial, the pressures in the system, and the quantities of radioactivity involved (Figure 4). As a complement, manually calculate the decay-corrected radiochemical yield (RCY) according to the formula:
   RCY =
   with residual activities describing the activities remaining in the reaction vial, on the C₁₈ cartridge, and in the waste vial at the end of the synthesis.

Figure 4: Typical distribution profile of radioactivity within the module. (A) reaction vial; (B) C₁₈ cartridge during synthesis of [⁶⁸Ga]Ga-3BP-3940. The flow of ⁶⁸Ga eluate into the reaction vial occurs at 6 min. The activity remains in the reaction vial throughout the radiolabeling reaction. After 16 min, the activity is transferred to the SPE cartridge. The cartridge is eluted after 19.5 min, after which a residual activity of around 150 MBq remains on the stationary phase. Please click here to view a larger version of this figure.

7. Dispensing and quality controls of [⁶⁸Ga]Ga-3BP-3940

1. Transfer the terminal vial to an appropriate shielded cell for radioactivity measurement and patient dose preparation.
2. Measure the activity of the terminal vial with a properly calibrated dose calibrator and record the preparation on the computer.
3. Identify the vial correctly and place it in an appropriate shielded container. Using appropriate aseptic techniques and radiation protection techniques, withdraw a ~0.5 mL sample from the terminal vial for quality controls.
   1. Assess the appearance of the preparation by visual inspection. Assess the pH of the preparation by depositing a drop of product solution on the pH paper strip.
   2. Measure the radiochemical purity by radio-TLC by depositing a drop of product solution on each of the two previously prepared iTLC-SG plates. Then, allow the plates to migrate into the corresponding mobile phases and read by the radiochromatograph. Integrate the resulting radiochromatogram by measuring the area under the curve of the product signal and the impurity signal, then calculate the radiochemical purity according to the formula:
      RCP (%) = 100 - %AUC_{impurity 1} - %AUC_{impurity 2}
   3. Measure the radiochemical purity by radio-HPLC by injecting ~50 µL of the preparation in the HPLC vial prepared beforehand (see step 2.3.7). Position the vial in the HPLC autosampler at the required position and start the analysis sequence. Once the sample has been injected into the system, remove the vial from the autosampler and replace it in its shielded container to minimize irradiation. At the end of the analysis, integrate the resulting radiochromatogram by measuring the area under the curve of the product signal and the impurities signals, then calculate the radiochemical purity according to the formula:
      ​RCP (%) = 100 - Σ(%AUC_impurities)
   4. Check the half-life (gamma counter) of the radioisotope contained in the preparation by adding ~5 µL of preparation to a gamma counter tube containing 1 mL of WFI. Count the tube with the gamma counter 10x consecutively (counts of 1 min each). Calculate the half-life based on the radioactive decay observed over the 10 measurements.
   5. Perform the radionuclidic identity assay on the same sample as the half-life assay by conducting gamma spectrometry analysis using the gamma counter, seeking the 511 keV and 1077 keV peaks from annihilation photons.
   6. Assess the radionuclidic purity after a 48 h decay period of the previous sample. Perform a 120 min measurement in the gamma counter, allowing the detection of any residual ⁶⁸Ga activity formed in situ from ⁶⁸Ge breakthrough and other long half-life radionuclide impurities.

8. Stability of the [⁶⁸Ga]Ga-3BP-3940 preparation

1. Assess three test batches for stability over time. For this purpose, withdraw approximately 200 µL of preparation from the terminal vial every hour immediately after the EoS and up to 4 h post-synthesis.
2. Perform a radio-HPLC analysis at each time point to measure RCP by HPLC according to the procedure described above. Perform Radio-TLC analyses at each timepoint to measure RCP by TLC according to the procedure previously described.

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 final molarity of 0.1 M was used. Methionine 10 mg/mL was added to the reaction medium as an anti-radiolysis agent to limit the degradation of the vector molecule during the heating step. Preliminary tests without terminal purification compared this antioxidant to gentisic acid 16 mg/mL and ascorbic acid 12 mg/mL and showed better final RCP with methionine than with either of these two other compounds (Figure 5).

Figure 5: HPLC radiochromatograms of crude reaction media from preliminary experiments comparing anti-radiolysis compounds. (A) Gentisic acid 16 mg/mL (RCP = 75.5%); (B) Ascorbic acid 12 mg/mL (RCP = 86.4%); (C) Methionine 10 mg/mL (RCP = 94.7%). Please click here to view a larger version of this figure.

To validate this automated radiolabeling method, three test batches were produced, resulting in preparations with an average final activity of 737 ± 2.8 MBq (generator at +2 weeks after calibration). Of note, activity losses in the single-use kit system during the synthesis were low, with a mean of 24.9% ± 3.4% over the three test productions (Figure 6), demonstrating the efficiency of the process.

Figure 6: Distribution of average residual activities for the three validation batches of [⁶⁸Ga]Ga-3BP-3940. The average proportion of total activity involved in the 3 test radiolabeling operations and found in the various elements of the system is detailed as a diagram, and the corresponding absolute activity values for each test synthesis are provided as a table. Please click here to view a larger version of this figure.

The three validation batches passed all quality controls, including radionuclide identification (Supplementary Table 1), calculated half-life (Supplementary Table 2), and radionuclide purity (Supplementary Table 3). In particular, excellent RCP values were obtained in both radio-TLC (mean RCP = 99.19 ± 0.07%; %CV = 0.07) (Supplementary Table 4) and radio-HPLC (mean RCP = 99.19 ± 0.07%; %CV = 0.07; Figure 7, Supplementary Table 5). The reproducibility of RCP values is excellent and may be further confirmed by the results of future syntheses. Also, the RCP values of the three test batches showed great stability over time, both in radio-TLC and radio-HPLC, with values consistently >98% up to 4 h after EoS (Figure 8, Supplementary Table 6). The comprehensive results of the analyses performed on these samples are compiled in Table 1. Overall, the automated ⁶⁸Ga radiolabeling method of 3BP-3940 was validated with a mean of 74.4% ± 3.3% decay-corrected RCY (calculated from RCP in HPLC). With the use of a pharmaceutical-grade vector, this method could be transposed unchanged to clinical use.

Figure 7: Representative radio-TLC. The results in (A) citrate buffer 0.1 M pH 4 and (B) ammonium acetate 1 M in 1:1 water/methanol. (C) Representative radio-HPLC spectra obtained when measuring the RCP of [⁶⁸Ga]Ga-3BP-3940. Please click here to view a larger version of this figure.

Figure 8: Stability results. Radiochemical purity of the three validation batches of [⁶⁸Ga]Ga-3BP3940 over time, from EoS to 4 h post-synthesis, determined by radio-HPLC. Please click here to view a larger version of this figure.

Table 1: Mains quality control results for three [⁶⁸Ga]Ga-3BP-3940 test batches.

Supplementary Figure 1: Supplies for the synthesis module setup. Please click here to download this File.

Supplementary Figure 2: Checklist for installing the kit on the synthesis module. Please click here to download this File.

Supplementary Figure 3: Alternative automated synthesis protocol with SCX cartridge. Please click here to download this File.

Supplementary Table 1: Radionuclide identification for the 3 test batches of [⁶⁸Ga]Ga-3BP-3940. Please click here to download this File.

Supplementary Table 2: Calculated half-life for the 3 test batches of [⁶⁸Ga]Ga-3BP-3940. Please click here to download this File.

Supplementary Table 3: Radionuclide purity for the 3 test batches of [⁶⁸Ga]Ga-3BP-3940. Please click here to download this File.

Supplementary Table 4: Radiochemical purity determined by radio-TLC for the 3 test batches of [⁶⁸Ga]Ga-3BP-3940. Please click here to download this File.

Supplementary Table 5: Radiochemical purity determined by radio-HPLC for the 3 test batches of [⁶⁸Ga]Ga-3BP-3940. Please click here to download this File.

Supplementary Table 6: Radiochemical stability over 4 h determined by radio-TLC for the 3 test batches of [⁶⁸Ga]Ga-3BP-3940. Please click here to download this File.

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 and straightforward and can be divided into three major phases: (i) transfer of all reagents into the reaction vial, (ii) radiolabeling of 3BP-3940 with ⁶⁸Ga, and (iii) product purification and formulation. Importantly, one of the great strengths of this automated method is its short duration, with the three test batches produced in 22.3 ± 0.6 min (filter integrity test time not included). In comparison, Hörmann et al., who reported on the automated synthesis of [⁶⁸Ga]Ga-FAP-2286 on a GRP-3V module, underlined a synthesis time of 35 min³⁸.

To ensure a radiolabeled product with high specific activity, only 30 µg of 3BP-3940 was used in the reaction, resulting in a mean specific activity of 24.3 ± 0.9 MBq/µg at EoS for the three test batches. Comparatively, average specific activities reported in the literature range from 6.9 MBq/µg³⁸ to 19.7 MBq/µg²⁹. The only exception is the [⁶⁸Ga]Ga-FAP-2286 preparation protocol described by Pang et al., which uses 25 µg of vector, resulting in an estimated specific activity of 40.7 MBq/µg (uncorrected for decay during the synthesis time)¹⁷.

Several automated protocols in the literature include an initial step of enrichment and purification of the gallium-68 eluate on a strong cation exchange (SCX) cartridge in order to control the volume of ⁶⁸Ga in an acidic solution added to the reaction medium⁴⁷. To shorten the preparation time and given the pharmaceutical grade of the GALLIAD ⁶⁸Ga generator, we decided to omit this step; consequently, the protocol described here is compatible with radiolabeling involving ~1.1 mL of ⁶⁸Ga eluate in 0.1 M HCl.

The type and molarity of the reaction buffer are crucial parameters in optimizing ⁶⁸Ga radiolabeling reactions⁴⁸. The challenge is to maintain a pH of approximately 3.6 to facilitate the chelation of the radiometal by the DOTA chelator while minimizing the formation of insoluble gallium hydroxides⁴⁹. Sodium acetate, at a final concentration of 0.07 M in the reaction medium, was retained based on its excellent performance in radiolabeling other vectors in our practice⁴⁶. Consistently, it demonstrated high efficiency in radiolabeling [⁶⁸Ga]Ga-3BP-3940 in this study. Moreover, this buffer is the most commonly reported in the literature for ⁶⁸Ga radiolabeling of FAP-targeting pseudopeptides, though typically used at higher molarities (0.25 M to 1 M)^{17,19,29,35,36}. A single article mentions the use of 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) as a reaction buffer³⁸. Its weak metal complexing properties make HEPES a very suitable compound for controlling the pH of ⁶⁸Ga radiolabeling reactions⁵⁰. However, regulatory constraints on this compound require additional tests to determine its residual quantities in the final radiopharmaceutical preparation, causing further delays between synthesis completion and product use^51,52.

Reported temperature settings for the preparation of ⁶⁸Ga-labeled FAP-inhibiting pseudopeptides vary widely in the literature. In preliminary works and in the corresponding scientific article^29,36, the team of Richard Baum observed increased by-product formation when radiolabeling reactions were conducted at temperatures exceeding 90 °C. In this context, lowering the heating temperature appeared as an effective strategy for minimizing the formation of degradation products. Of note, no antioxidant compounds were added to the reaction mixture under these conditions. Nevertheless, the most commonly reported conditions in the literature therefore include heating at 85 °C for 15 min. Even lower heating temperatures can be found, with the ⁶⁸Ga radiolabeling conditions of a close analog of 3BP-3940, additionally functionalized with a fluorescent group, involving 80 °C heating for 15 min⁵³. Conversely, two protocols for the automated preparation of [⁶⁸Ga]Ga-FAP-2286 mentioned a heating temperature of 120 °C for 10 min⁵⁴ and 125 °C for 6 min³⁸, respectively, but resulting in the appearance of by-products for the second. We therefore opted for an intermediate temperature, heating at 98 °C for 8 min, similar to some published processes^19,35. Based on the RCP of the three test batches, heating under these conditions in the presence of an antioxidant does not appear to result in the significant formation of radiolysis products during labeling.

Even though it is not systematically used, the addition of an antioxidant compound to the reaction medium can enhance the radiolabeling outcome by achieving higher RCP⁴³. In the final radiopharmaceutical formulation, an antioxidant compound can limit radiolysis phenomena and improve the stability of the radiocomplex over time^55,56,57. In the case of ⁶⁸Ga-labeled, FAP-targeting DOTA-pseudopeptides, some studies suggested insufficient efficacy of ascorbic acid (10 mg or 50 mg in a reaction volume of ~2.6 mL) in preventing the formation of radiolysis by-products³⁸. Interestingly, forced degradation tests in the presence of hydrogen peroxide showed an increase in the proportion of these impurities, suggesting oxidation products, probably formed through the transformation of one or more sulfide groups of the pseudopeptide into sulfoxides and/or sulfones⁵⁸. This underlines the importance of antioxidants in the reaction medium for the radiolabeling of 3BP-3940 and related molecules. In the present work, preliminary assays (see Figure 5) have identified methionine as an ideal compound for this specific purpose. Continuing with the above hypothesis, it is possible that under oxidizing conditions, sulfone and/or sulfoxide derivatives are preferentially formed with methionine, thereby efficiently preserving the integrity of the sulfide functions in 3BP-3940. In addition to its use in the automated ⁶⁸Ga radiolabeling protocols of various other vectors labeled with ⁶⁸Ga^59,60 or ¹⁷⁷Lu^56,61,62,63, methionine is justifiably reported to be used in the preparation of [⁶⁸Ga]Ga-3BP-3940³⁵ and [⁶⁸Ga]Ga-FAP-2286¹⁹, in combination with ascorbic acid. Notably, in this protocol, the volume and concentration of each reagent (buffer and antioxidant) were selected to achieve the optimal reaction pH without requiring prior adjustment with concentrated HCl, as described in some methods^47,48,64. Again, accurate control of the reaction pH is a key parameter for successful gallium-68 radiolabeling.

The final purification step by solid-phase extraction is necessary to remove traces of free ⁶⁸Ga³⁺ that have not been complexed with 3BP-3940. As the C₁₈ cartridge used in the assays showed good results, with an excellent recovery rate (80.3% ± 3.3% of the activity bound to the cartridge recovered after elution), other models were not tested. Interestingly, water-wettable hydrophilic-lipophilic balanced (HLB) reverse-phase cartridges are also reported for the terminal purification of [⁶⁸Ga]Ga-3BP-3940^29,36.

As it stands, the primary limitation of this protocol is its compatibility with only a single model of ⁶⁸Ga generator. However, solutions can be considered for adapting the method to other generators, such as increasing the buffer volume proportionally to the volume of ⁶⁸Ga eluate. For instance, using 1.25 mL instead of 0.25 mL of 0.8 M sodium acetate buffer could be suitable for a 5 mL ⁶⁸Ga eluate. Another approach would involve incorporating an SCX cartridge into the setup (typically between manifolds A2 and B1) to concentrate the ⁶⁸Ga³⁺ ions from the generator eluate. Then, gallium could be recovered in a fixed volume by eluting the SCX cartridge with a saturated solution of NaCl (5 M) at pH 1. This modification also enables multi-generator syntheses, as demonstrated by Mueller et al., who employed up to four generators for the preparation of [⁶⁸Ga]Ga-FAP-2286^19,35. Thus, an alternative protocol incorporating an SCX cartridge is proposed in Supplementary Figure 3. Such an approach would allow a significant upscaling of the method, and could greatly increase the number of patients who could benefit from a given preparation. However, in the method, it is essential to note that buffer volumes should once again be carefully adjusted to allow, under these alternative conditions, a reaction pH of around 3.6, ideal for ⁶⁸Ga radiolabeling⁶⁵. The anti-radiolysis agent and buffer should also be prepared directly in the same syringe at B3 to make the C1 position free for the SCX cartridge eluent.

Lastly, as the automated process presented here is GMP-compliant, the use of GMP-grade 3BP-3940 in association with the development and validation of additional quality controls (i.e., preparation sterility, bacterial endotoxins levels, radiochemical identity by radio-HPLC and residual solvent content quantification) would ensure a straightforward transition to clinical applications. This implementation would obviously have to meet all current regulatory requirements to be validated, in particular, the constitution of an IMPD.

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 thank Yasmine Soualy, Stéphane Renaud and Élodie Gaven for their help in preparing the radiolabeling reactions presented in this manuscript.