An Automated Radiosynthesis of [⁶⁸Ga]Ga-FAPI-46 for Routine Clinical Use

Abstract

[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.

Introduction

Fibroblast activation protein (FAP) has become a prominent target for cancer imaging and therapy^1,2. 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 achieved^4,5,6. 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), Germany^7,8,9,10,11, drawing on earlier work identifying the highly promising N-(4-quinolinoyl)-glycyl-(2-cyanopyrrolidine) scaffold for FAP inhibition^12,13. 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 arthritis^19,20. 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-04^6,10. 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-04^23,24. 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 generators^{26,27,28,29,30,31,32,33} 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 years^35,36.

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 radiopharmaceuticals^37,38,39. 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.

Protocol

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

1. Turn on the Hot cell compressed air, nitrogen gas supply, ventilation, light, and power to the laptop and programmable logic controller (PLC). Check the level of the module waste bottle. Replace with an empty bottle when ¾ full.
2. Turn the computer on and log on to the synthesizer software. Enter the username and password.
3. On the synthesizer software, press Download recipe, select the Excel sequence file for the production of [⁶⁸Ga]Ga-FAPI-46, and press Open. Press OK after the sequence has downloaded successfully.
4. Press Start; input the batch number, reagent kit lot number, and any comments in the pop-up window; and press OK.
   NOTE: From that point on, the user interface will display a step message describing the step action and/or the prompting operator for intervention.
5. Remove the old hardware kit as directed by the step message on the user interface. Press NEXT on the user interface.
6. Remove the generator elution syringe (HCl Syringe 1) from the synthesizer, as directed by the step message on the user interface, by pulling the thumb rest of the syringe out of the syringe driver slot and unscrewing the syringe from the generator inlet line. Press NEXT on the user interface.

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.

1. Obtain the reagent kit and ancillaries set, a 50 mL centrifuge tube containing a minimum of 5 mL of water with trace level concentration of metals; the HCl 0.1 M bag, 7 mg of ascorbic acid vial (vial A); the sodium ascorbate vial (5 mg) (Vial B); 2 x 5 mL polypropylene (PP)/polyethylene (PE) syringes, free of latex, PVC and silicone oil (three if using dual generator elution) and the 50 µg FAPI-46 glass vial (Vial C; see Supplemental Figure S1A,B-Supplemental File 1).
2. Pour approximately 2 mL of water into the cap of the 50 mL tube. Draw 1 mL of water in a 5 mL syringe (label Syringe A). Cap with a needle. Pipette 400 µL of water and add to the 7 mg ascorbic acid vial (vial A) - gently shake to dissolve.
3. Open the provided 0.25 M sodium acetate buffer vial (from the reagent kit) and 50 µg FAPI-46 glass vial. Pipette 0.9 mL of buffer and add to the 50 µg FAPI-46 glass vial (vial B); mix gently.
4. Withdraw the contents of Vial A and Vial B into Syringe A. Mix gently.
5. Label the 5 mL syringe provided in the ancillaries set as Syringe B. Withdraw 5 mL of 0.1 M HCl from the 0.1 M HCl bag with Syringe B; cap with the provided dispensing pin.
   NOTE: For production using dual generator elution, two syringes of 5 mL of 0.1 M HCl are required.
6. Label the 3 mL syringe provided in the ancillaries set as Syringe C. Open the acidified 5 M sodium chloride solution vial (from the reagent kit), pipette 1 mL, transfer into Syringe C, and cap with the provided dispensing pin.
7. Withdraw 1 mL of 0.9% saline into a 5 mL syringe (Syringe D) and add to vial C; keep Syringe D empty.

3. Preparation of the synthesis cassette and cassette installation

1. Obtain a dedicated sterile [⁶⁸Ga]Ga cassette.
2. Assemble the cassette following the steps below:
   1. Unwrap the cassette envelope, check for any damage, and tighten each Luer connection. Rotate and align each stopcock on the cassette to ensure they are not stuck and will fit on the module. Remove all the spike caps.
   2. Condition the strong cationic exchange prepurification SPE cartridge as follows:
      1. Remove the cartridge positioned on Manifold 3 (M3) valve 7 from the cassette.
      2. Place an open dedicated glass waste bottle on the bench.
      3. Attach the syringe containing 2 mL of 3 M HCl syringe (provided in the reagent kit) onto the SPE cartridge and slowly elute the SPE cartridge dropwise into the glass waste bottle. Remove the syringe, withdraw 5 mL of air, and flush the SPE with 5 mL of air.
         CAUTION: Step 3.2.2.3 requires the handling of a strong acid (3 M hydrochloric acid). Wear appropriate protective equipment (gloves, safety glasses, lab coat).
      4. Attach the syringe containing 5 mL of water (from the reagent kit) to the SPE and slowly elute the SPE dropwise into the glass waste bottle. Remove the syringe, withdraw 5 mL of air, and flush the SPE with 5 mL of air.
      5. Place the SPE back on the manifold in position M3 valve 7.
   3. Press NEXT on the user interface.
   4. Assemble and install the new cassette as shown on the user interface (see Figure 1A,B if using dual elution), without any reagents, and following the connections described in Figure 1C
      1. Connect the 10 mL syringe (from the ancillaries set) to M2 valve 6.
      2. Install the four manifolds and lock the cassette using the magnetic locks of the synthesizer.
      3. Place the reactor in the oven.
      4. Connect tubings to G1, W2, W1, R, and G2 ports.
      5. Connect the M1 valve 3 right side tubing to the cation exchange cartridge on M3 valve 7.
      6. Install an SPE postpurification cartridge on M2 valve 4.
         NOTE: A photo of the final setup for the synthesis is shown in Supplemental Figure S1C (Supplemental File 1).
3. Press NEXT on the user interface to initiate the following tests in the sequence listed below:
   1. Pressure testing of inert gas connection to M4.
   2. Pressure testing connection between M3 and M4.
   3. Pressure testing the reactor.
   4. Pressure testing the waste connection to M3.
   5. Pressure testing the postpurification SPE cartridge between M3 and M2.
   6. Pressure testing the syringe connection on M2 valve 6.
   7. Pressure testing the waste connection to M2.
   8. Pressure testing of inert gas connection to M1.
   9. Pressure testing the prepurification SPE cartridge between M1 and M3.
   10. Flushing the spikes on valves 10, 11, and 12 with inert gas.
   11. Flushing M3 and M4 with inert gas.
   12. Flushing both prepurification and postpurification SPE cartridges with inert gas.

4. Reagents, generator(s) line(s), and final product vial Installation (see Figure 1A,C and Figure 1B,C if using dual generator elution)

1. Obtain the ethanol (100%), saline, and water for injection vials from the reagent kit; remove the caps; and swab each septum with an alcohol wipe.
2. Install the vials on the kit at positions M4 valve 10, M4 valve 11, and M4 valve 12 when prompted by the synthesizer; click NEXT on the user interface.
3. Remove the needle from Syringe A and draw the plunger to 5 mL.
4. Disconnect the line to the reactor middle port and transfer the content of Syringe A into the reactor vial via the reactor middle port.
5. Re-connect the line to the reactor middle port; click NEXT on the user interface.
6. Install the generator outlet line(s) on the cassette at position M1 valve 2 (and M1 valve 1 if dual generator elution); click NEXT on the user interface.
7. Remove the dispensing needle from Syringe C and install it at position M1 valve 3; click NEXT on the user interface.
8. Remove the dispensing needle from Syringe B, connect Syringe B luer lock on the generator inlet line fitting, and push Syringe B so the thumb rest of the plunger slides into the syringe driver slot; click NEXT on the user interface.
9. Prepare the final product vial in a Class II Biological safety cabinet or equivalent, following the steps below:
   1. Place a labeled 25 mL sterile vial in a tungsten pot (or suitably shielded pot).
   2. Swab the septum of the vial with an alcohol wipe, place a lid on the pot, and insert a filtered vent needle.
   3. Connect the outlet of a low protein-binding sterilizing 0.22 µm polyvinyl difluoride (PVDF) vented filter to a sterile 20 G hypodermic needle. Label the filter with the product batch number.
   4. Insert the needle into the vented 25 mL final product vial.
   5. Withdraw the content of Vial C (Table 1) into empty Syringe D (see step 2.7) and add 4 mL of air.
   6. Connect Syringe D to the inlet of the 0.22 µm vented filter. Push the syringe contents through the 0.22 µm filter and needle into the final product vial.
   7. Flush the filter 2x with 5 mL of air. Remove the syringe from the 0.22 µm vented filter.
      NOTE: The flushes should be done slowly to avoid rupturing the filter membrane.
10. Transfer the pot containing the final product vial to the hot cell, connect the end tubing from position M2 valve 5 to the product vial filter, and click NEXT on the user interface.

5. Synthesizer preliminary steps prior to radiolabeling

1. Wait for the following preliminary steps to be performed by the synthesizer: pressurizing ethanol vial (M4, valve 10); pressurizing saline vial (M4, valve 11); pressurizing water vial (M4, valve 12); pressurizing the acidified sodium chloride syringe (M1, valve 3); conditioning the postpurification SPE cartridge with ethanol; conditioning the postpurification SPE with water; repressurizing the water vial (M4, valve 12).
2. Press NEXT on the user interface to start the production when prompted by the synthesizer Ready to elute ⁶⁸Ga Generator.

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.

1. At the end of synthesis (EOS), look for the message synthesis complete displayed by the synthesizer. Remove the sterilizing 0.22 µm vented filter and the filtered vent needle from the final product vial and retrieve the tungsten pot from the hot cell.

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

1. Transfer the product vial to an appropriately shielded dispensing system and invert the vial to homogenize the product.
2. Using aseptic techniques and radiation protection techniques, withdraw a 1 mL sample from the product vial and:
   1. Aliquot 150 µL into a low protein-binding 1.5 mL tube (Tube 1) for prerelease quality control testing + residual solvent analysis.
   2. Aliquot 50 µL into a low protein-binding 1.5 mL tube (Tube 2) for endotoxin testing.
   3. Aliquot 300 µL in a 10 mL sterile evacuated glass vial for sterility testing (send to an external contractor after the sample has decayed to an acceptable level [10 half-lives]).
   4. Aliquot 500 µL in a 10 mL sterile evacuated glass vial for retention.
   5. Aliquot the remaining product in a vial (~10 mL) for patient doses.

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.

1. Assess the appearance by visual inspection.
2. Assess the pH with an indicator paper.
3. Determine the half-life of the radionuclide by taking three activity measurements of a 50 µL sample, using an ionization chamber.
4. Assess radionuclidic identity and radionuclidic purity using a multichannel analyzer (MCA)-based NaI(Tl) gamma spectrometer.
5. Assess the radiochemical identity with analytical radio-HPLC by verifying that the retention times of the [⁶⁸Ga]Ga-FAPI-46 sample and a non-radioactive [^natGa]Ga-FAPI-46 reference standard are similar (allowing for a small delay volume between the radioactivity and UV detectors).
6. Quantify radiochemical % purity with analytical radio-HPLC using equation (1).
    
7. Quantify radiochemical % purity with analytical radio-TLC using equation (2).
    
   NOTE: Steps 8.8, 8.9, and 8.10 may be performed pre- or post- release based on local regulatory requirements. Steps 8.11 and 8.12 may be performed post release. Any post release testing should be performed as soon as practicable after radioactive decay of the sample.
8. Quantify residual solvent content (ethanol) of the formulation using gas chromatography.
9. Assess bacterial endotoxin levels using a cartridge-based endotoxin testing system (kinetic chromogenic limulus amebocyte lysate [LAL] method).
10. Assess the integrity of the 0.22 µm filter by performing a bubble test.
11. Assess radionuclidic purity (germanium-68 breakthrough) using a gamma spectrometer or a gamma counter. Retain the solution to be examined for at least 48 h to allow the gallium-68 to decay to a level that permits the detection of impurities.
12. Assess the sterility of the product.

9. Stability testing

1. For stability analysis, withdraw 50 µL of [⁶⁸Ga]Ga-FAPI-46 from the final product vial immediately after EOS, and then again at 1 h intervals up to 3 h post EOS.
2. Quantify radiochemical % purity via radio-HPLC at each time point.
3. Quantify radiochemical % purity via radio-TLC at each time point.

Representative Results

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 TLC for 5, 10, 15, and 20 min of reaction time (n = 3). Abbreviations: TLC = thin layer chromatography; Exp. = experiment. Please click here to download this Table.

Table 3: Summary of the % recovery and TLC and HPLC radiochemical purity analyses of [⁶⁸Ga]Ga-FAPI-46 at EOS, using four different solid phase extraction cartridges (n = 3). Abbreviations: SPE = solid phase extraction; FAPI = fibroblast activation protein inhibitor; EOS = end of synthesis; RSD = relative standard deviation; TLC = thin layer chromatography; HPLC = high-performance liquid chromatography. Please click here to download this Table.

The automated protocol for the production of [⁶⁸Ga]Ga-FAPI-46 was developed using the 10 min reaction time and the Strata X 60 mg SPE cartridge. Two series of three validation batches were performed using single generator and dual generator elution(s), yielding average final product activities of 0.60 GBq and 1.6 GBq, respectively (Table 4). In both series, the final [⁶⁸Ga]Ga-FAPI-46 product passed all quality control tests (Table 4). The stability of the [⁶⁸Ga]Ga-FAPI-46 product was evaluated by assessing the radiochemical purity of the product by TLC and HPLC analysis (Figure 3 and Figure 4) for all six productions for up to 3 h post synthesis and was greater than 98.0 ± 1.8% (n = 3) and 99.1 ± 0.9% (n = 3), respectively. Overall, the synthesis of [⁶⁸Ga]Ga-FAPI-46 was validated in 89.8 ± 4.8% decay corrected yield (n = 6). The automated production is currently being used for the production of [⁶⁸Ga]Ga-FAPI-46 to support clinical trials.

Table 4: Summary of the QC results for [⁶⁸Ga]Ga-FAPI-46 validation runs (n = 6): at low activity from a single generator elution (n = 3) and high activity from a dual generator elution (n = 3). Please click here to download this Table.

Figure 3: TLC chromatogram of [⁶⁸Ga]Ga-FAPI-46: (Application of 5 µL) at EOS using iTLC-SG paper and 1 M ammonium acetate/methanol (1:1 v/v) eluent. The radioactivity was measured using a radio-TLC scanner equipped with a PS Plastic radio detector, integrating at each 1 mm increment at a rate of 1 mm/s. Abbreviations: TLC = thin layer chromatography; FAPI = fibroblast activation protein inhibitor; EOS = end of synthesis; iTLC-SG = instant TLC-silica gel. Please click here to view a larger version of this figure.

Figure 4: HPLC analysis of [⁶⁸Ga]Ga-FAPI-46 final product. HPLC RP-18 encapped Column, mobile phase A: 0.1% trifluoroacetic acid, mobile phase B: neat acetonitrile. Gradient: 0-0.5 min 2% B, 0.5-10.0 min 2%-35% B, 10.0-12.0 min 35% B, and 12.0-15.0 35% B. The UV wavelength was 280 nm and flow rate 2 mL/min. (A) UV-chromatogram of [⁶⁸Ga]Ga-FAPI-46 product including the UV signal for sodium ascorbate at 1.0 min and the two small peaks at 4.5 and 4.8 min corresponding to [^natGa]Ga-FAPI-46 and FAPI-46 precursor, respectively. The UV-chromatograms of 10 mg/L [^natGa]Ga-FAPI-46 standard and 10 mg/L FAPI-46 precursor are shown in Supplemental Figure S2 (Supplemental File 1). (B) Radiochromatogram of [⁶⁸Ga]Ga-FAPI-46 (Rt [⁶⁸Ga]Ga-FAPI-46 = 4.6 min - Rt free [⁶⁸Ga]Ga³⁺ = 1.1 min). Abbreviations: HPLC = high-performance liquid chromatography; FAPI = fibroblast activation protein inhibitor; Na⁺Asc^- = sodium ascorbate. Please click here to view a larger version of this figure.

Supplemental File 1: This document includes: Supplemental Table S1 - Reported automated syntheses and quality control details for [⁶⁸Ga]Ga-FAPI-46, Supplemental Section 1 - Preliminary experiments: Optimization of the reaction time and selection of postpurification SPE cartridge for the automated production of [⁶⁸Ga]Ga-FAPI-46 on the MS radiosynthesizer, Supplemental Figure S1 - Images of reagents and final set-up for automated synthesis of [⁶⁸Ga]Ga-FAPI-46 on MS synthesizer, Supplemental Figure S2 - UV-chromatograms of [^natGa]Ga-FAPI-46 and FAPI-46 precursor, and Supplemental Figure S3 - Radio-chromatogram of [⁶⁸Ga]Ga-FAPI-46 for high activity radiolabeling (2.8 GBq) without ascorbic acid in the reactor. Please click here to download this File.

Discussion

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]Ga-FAPI-46 was going to be developed, these preliminary experiments were also conducted using the MS synthesizer where possible. A [⁶⁸Ga]Ga-FAPI-46 sequence was created using similar parameters used for the production of [⁶⁸Ga]Ga-PSMA-11 and [⁶⁸Ga]Ga-DOTA-TATE. The sequence is based on simple stepwise programming in an Excel spreadsheet and can be easily edited. The kits and reagents were assembled for the first part of the [⁶⁸Ga]Ga-FAPI-46 synthesis only (⁶⁸Ga elution and purification; labeling reaction), omitting the final steps (SPE purification and final sterile filtration) as these preliminary experiments were performed on the crude material manually. As a result, the post-purification SPE cartridge (Strata X 60 mg) as well as the ethanol, water, and saline vials on Manifold 4 and the final product vial did not need to be installed.

The published literature on the production of [⁶⁸Ga]Ga-FAPI-46 describes the purification of the final product using different SPE cartridges: OASIS HLB 30 mg sorbent^23,29, OASIS HLB Plus 225 mg²⁸, CM^26,34, C18 Plus Sep-Pak^30,32, and Sep-Pak Light C18³¹. This work compares three of the different SPE cartridges used in the literature (HLB 30 mg; C18 SepPak Plus Short, and HLB 225 mg) with the Strata X 60 mg that is provided with our existing cassettes for the purification of [⁶⁸Ga]Ga-PSMA-11 and [⁶⁸Ga]Ga-DOTA-TATE. To the best of our knowledge, the Strata X 60 mg SPE cartridge had not yet been evaluated for [⁶⁸Ga]Ga-FAPI-46 purification. The Strata X 60 mg showed either similar (HLB 30 mg; C18 SepPak Plus Short) or better (HLB 225 mg) performance than other SPE used in the literature for the purification of [⁶⁸Ga]Ga-FAPI-46, with respect to trapping and elution efficiency (Table 3). Elution of the HLB 225 mg with an extra milliliter of ethanol did not result in a significant improvement (results not shown). The Strata X 60 mg cartridge was selected for validation of the fully automated synthesis described in this protocol.

The automated synthesis of [⁶⁸Ga]Ga-FAPI-46 outlined in the above protocol can be described in four main steps. The first step is the elution of one or two ⁶⁸Ge/⁶⁸Ga generators with 0.1 M HCl and subsequent trapping of [⁶⁸Ga]GaCl₃ on the ion exchange cartridge. This is followed by the elution of [⁶⁸Ga]Ga³⁺ from the ion exchange cartridge into the reaction vessel (preloaded with the precursor solution containing 50 µg of FAPI-46 precursor, 1.4 mL of water containing trace level concentration of metal, and 7 mg of ascorbic acid) using an acidified 5 M sodium chloride solution. The pH of the labeling reaction is 3.5-4. The next step is the radiolabeling reaction during which the reactor is heated at 95 °C for 10 min to allow complexation of the [⁶⁸Ga]Ga³⁺ by the DOTA moiety of the FAPI-46 precursor. After cooling of the reaction mixture to 40 °C, the fourth and final step is the purification of [⁶⁸Ga]Ga-FAPI-46 consisting of (i) trapping and purification of the reaction mixture on the post purification Strata X 60 mg SPE cartridge, (ii) rinsing of the Strata X 60 mg SPE cartridge with water for injection, (iii) elution of the final product with ethanol, (iv) dilution with saline for injection, and (v) transfer into the final product vial through a 0.22 µm sterilizing vented filter. The reaction can be monitored in real time by visual inspection, and sensor readings (e.g., temperature, pressure, vacuum, radiation reading, and countdown timers). A representative screenshot of the user interface is shown in Figure 1A,B. The radioactivity profile shown in Figure 2 is particularly useful to monitor the progress of the synthesis by displaying the transfer of radioactivity through the critical steps described above.

Six validation runs passing all quality control specifications were performed, using either one or two ⁶⁸Ge/⁶⁸Ga generators (2.41 GBq). Three runs were completed using ⁶⁸Ga from a single generator elution, yielding 0.59-0.63 GBq of [⁶⁸Ga]Ga-FAPI-46. Three runs were completed using ⁶⁸Ga eluted from two generators, yielding 1.54-1.71 GBq of [⁶⁸Ga]Ga-FAPI-46. Each run was completed in ~25 min. The single generator method was designed anticipating the need for higher activities to be produced in the future. Consequently, 7 mg of ascorbic acid stabilizer was added to the reaction mixture to avoid radiolysis during the radiolabeling step, as described previously by Mu et al. for the production of [⁶⁸Ga]Ga-DOTA-TATE⁴¹. Da Pieve et al.²⁹ and Alfteimi et al.³⁰ have also reported the addition of 0.3 mg ascorbic acid stabilizer to the reaction mixture for the automated production of [⁶⁸Ga]Ga-FAPI-46 (Supplemental Table S1-Supplemental File 1). Indeed, we observed significant radiolysis for a high activity synthesis (~2.8 GBq in reactor) when the ascorbic acid stabilizer was omitted from the reaction mixture (Supplemental Figure S3-Supplemental File 1). In addition, to avoid radiolysis of the final product, 5 mg of sodium ascorbate in 1 mL of 0.9% saline for injection was added manually to the final product vial through the sterilizing filter, resulting in a final product volume of 11 mL. Each batch was tested up to 3 h post production and both the low activity (single generator elution) and high activity (dual generator elution) productions demonstrated excellent radiochemical stability (>95% by HPLC and TLC). It is important to emphasize that implementation of this protocol involves the handling of unsealed radioactive materials and should only be undertaken by appropriately trained staff. The protocol should be performed behind radiation shielding, preferably inside a ventilated hot cell. The expected purpose of the synthesis of [⁶⁸Ga]Ga-FAPI-46 using this protocol is administration to patients for diagnostic imaging. As such, strict aseptic techniques should be followed throughout. Quality control testing of [⁶⁸Ga]Ga-FAPI-46 as described in the above protocol is critical to ensure the quality of the final product.

It is anticipated that any site manufacturing [⁶⁸Ga]Ga-PSMA-11 or [⁶⁸Ga]Ga-DOTA-TATE on the MS synthesizer should be fully equipped to prepare [⁶⁸Ga]Ga-FAPI-46 using the protocol described here, with only minor modifications required for use of alternative generators or reagents. Due to the increasing demand for [⁶⁸Ga]Ga-FAPI-46 in clinics, the work presented here was designed to meet various site needs and capabilities, from the production of 1-2 patient doses of [⁶⁸Ga]Ga-FAPI-46 per synthesis on the single generator method and up to 4-5 patients per synthesis for the dual generator method, depending on PET camera(s) availability.

Materials

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