A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This work describes the automated production of up to 1.7 GBq of [68Ga]Ga-FAPI-46 on the iPHASE MultiSyn synthesizer for PET imaging of fibroblast activation protein.

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 therapy1,2. 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 achieved4,5,6. 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), Germany7,8,9,10,11, drawing on earlier work identifying the highly promising N-(4-quinolinoyl)-glycyl-(2-cyanopyrrolidine) scaffold for FAP inhibition12,13. 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 arthritis19,20. 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-046,10. 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-0423,24. 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 generators26,27,28,29,30,31,32,33 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 years35,36.

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 radiopharmaceuticals37,38,39. 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-protocol-12592
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-protocol-13561
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).
    figure-protocol-16198 
  7. Quantify radiochemical % purity with analytical radio-TLC using equation (2).
    figure-protocol-16409 
    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.

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 T...

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...

Disclosures

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.

Acknowledgements

The authors acknowledge and thank SOFIE Biosciences Inc. for supplying the FAPI-46 chemical precursor and [natGa]Ga-FAPI-46 standard, the Charlies Foundation for Research for financial support, Stan Poniger from iPHASE Pty Ltd, and the Radiopharmaceuticals Production team (RAPID) at the Medical Technology and Physics Department at Sir Charles Gairdner Hospital for their scientific and technical support. The authors also acknowledge the assistance of the WA National Imaging Facility Node, which is supported by infrastructure funding from the Western Australian State Government in partnership with the Australian Federal Government, through the National Collaborative Research Infrastructure Strategy (NCRIS) capability.

Materials

NameCompanyCatalog NumberComments
0.1 M Hydrochloric acid (HCl) ultra pureABX advanced biochemical compound (Radberg, Germany)HCl-103-GUsed for generator(s) elution
Ammonium acetateSigma Aldrich Pty Ltd (NSW, Australia)A1542-250GUsed to make iTLC mobile phase
C18 SepPak Plus short (360 mg)WatersWAT020515Post-purification silica SPE
Chromolith Performance RP-18 endcapped 100-4.6 monolithicMerck Pty Ltd (Victoria (Australia)1021290001HPLC RP-18 endcapped column, used for HPLC quality control
Dose calibratorCapintecCRC-15PETUsed to calibrate and measure 68Ga activity
Dual scan-RAM LabLogic Limited (VA, USA)SR-1ARadio-TLC scanner to analysise the iTLC paper
FAPI-46 precursor (GMP)ABX advanced biochemical compound (Radberg, Germany)3601.0000.050Peptide precursor
Fill ease Sterile vacuum vial (10 mL)HUAYI iosotopesSVV-10CUsed for sterility and retention samples
Fill ease Sterile vacuum vial (25 mL)HUAYI iosotopesSVV-25AUsed for final product
Ga68 peptide radiolabelling with generator pre-purificationiPHASE Technologies (Melbourne, Australia)MSR-120G-(RK-3296)Reagent set 
Ga68 radiolabeling with generator prepurificationiPHASE Technologies (Melbourne, Australia)MSH-120Hardware Cassette + ancillaries set 
Gas chromatography (GC) systemAgilent technologies (Vic, Australia)G2630AUsed to measure residual solvent
GS Standard source (Ba133)Global Medical Solutions Pty Ltd (Australia)D-102-19Used to calibrate the Gamma Spectometer
GS Standard source (Co60)Global Medical Solutions Pty Ltd (Australia)1559-84Used to calibrate the Gamma Spectometer
High performance liquid chromatography (HPLC) systemShimadzu Scientific Instruments (NSW, Australia)LC-20HPLC equipment
Hydrophobic air vent needleBaldwin Medical (Victoria, australia)1088Used with final product vial
 IGG100 Eckert & Ziegler Isotope Products  (Berlin, Germany)IGG100-65M-NT68Ge/68Ga generator
5 mL syringe (Injekt luer lock solo syringe)B Braun (Melsungen, Germany)4606710VPolypropylene (PP)/polyethylene (PE) syringes, free of latex, PVC, and silicone oil free syringe used for reagents
iTLC-SG paperAgilent technologies (Vic, Australia)SGI0001Used to for iTLC analysis
LabLogic software (LAURA)LabLogic Limited (VA, USA)LAURA software version 6.1Used to for radio-TLC analysis
L-Ascorbic acid Trace selectFluka Sigma05878-100GUsed as a radical scavenger in the reaction mixture
Lichrosolv Acetonitrile (ACN)Sigma Aldrich Pty Ltd (NSW, Australia)1.00030.2500Used to make HPLC organic mobile phase
Lichrosolv WaterSigma Aldrich Pty Ltd (NSW, Australia)1.15333.2500Used to make HPLC aqueous mobile phase
Methanol (MeOH)Sigma Aldrich Pty Ltd (NSW, Australia)1060182500Used to make iTLC mobile phase
Na+I- detectorLabLogic Limited (VA, USA)1"NaI / PMTRadiodetector used for radio-HPLC
Oasis HLB (30 mg)Waters (Milford, MA, USA)WAT094225Postpurification copolymer SPE
Oasis HLB Plus short (225 mg)Waters (Milford, MA, USA)186000132Postpurification copolymer SPE
pH stripsThermo Fisher Scientific Australia Tty Ltd90424Used to measure product pH
PS  detectorLabLogic Limited (VA, USA)PS plastic/PMTRadiodetector used for radio-TLC
 Safe Lock tube (1.5 mL)Eppendorf0030 120.086Used for quality control samples
(+)-Sodium L-ascorbateMerck Pty Ltd (Victoria (Australia)11140-250GStabilizer of the final product
Sodium chloride (NaCl) solution (saline)PfizerPS1110.9%, for injection, USP grade
Sterican 100 NeedlesB Braun (Melsungen, Germany)4667093Used for final product
Sterile syringe filter (0.22 µm)Millipore Sigma (Burlington, MA, USA)SLGSV255FMillex-GV
Strata SCX (in Hardware cassette kit)Phenomenex inside hardware kit from iPHASE Technologies (Melbourne, Australia)MSH-120Prepurification silica SPE inside Hardware Cassette
Strata X (in Hardware cassette kit)Phenomenex inside hardware kit from iPHASE Technologies (Melbourne, Australia)MSH-120Postpurification silica SPE inside Hardware Cassette
Trace Select Water for trace analysisHoneywell Riedel-de-Haen95305-2.5LUsed for reaction mixture and to precondition the prepurification SPE cartridge
Trifluoracetic acid (TFA)Sigma Aldrich Pty Ltd (NSW, Australia)302031-10X1mLUsed to make HPLC aqueous mobile phase
Ultra Fine insulin syringe (0.5 mL)BD326769Used for dispensing quality control samples
Vented filter Cathivex-GV 0.22 µm, low protein binding Durapore PVDF membraneMerk Millipore (Cork, Ireland)SLGV02505Used to filter the final product

References

  1. Imlimthan, S., et al. New frontiers in cancer imaging and therapy based on radiolabeled fibroblast activation protein inhibitors: a rational review and current progress. Pharmaceuticals. 14 (10), 1023 (2021).
  2. Siveke, J. T. Fibroblast-activating protein: targeting the roots of the tumor microenvironment. J Nucl Med. 59 (9), 1415-1423 (2018).
  3. Koczorowska, M. M., et al. Fibroblast activation protein-α, a stromal cell surface protease, shapes key features of cancer associated fibroblasts through proteome and degradome alterations. Mol Oncol. 10 (1), 40-58 (2016).
  4. Altmann, A., Haberkorn, U., Siveke, J. The latest developments in imaging of fibroblast activation protein. J Nucl Med. 62 (2), 160-167 (2021).
  5. Lindner, , et al. Targeting of activated fibroblasts for imaging and therapy. EJNMMI Radiopharm Chem. 4, 16 (2019).
  6. Linder, T., Giesel, F. L., Kratochwil, C., Serfling, S. E. Radioligands targeting fibroblast activation protein (FAP). Cancers. 13 (22), 5744 (2021).
  7. Loktev, A., et al. A tumor-imaging method targeting cancer-associated fibroblast. J Nucl Med. 59 (9), 1423-1429 (2018).
  8. Lindner, T., et al. Development of quinoline-based theranostic ligands for the targeting of fibroblast activation protein. J Nucl Med. 59 (9), 1415-1422 (2018).
  9. Giesel, F. L., et al. 68Ga-FAPI PET/CT: biodistribution and preliminary dosimetry estimate of 2 DOTA-containing FAP-targeting agents in patients with various cancers. J Nucl Med. 60 (3), 386-392 (2019).
  10. Loktev, A., et al. Development of fibroblast activation protein-targeted radiotracers with improved tumor retention. J Nucl Med. 60, 1421-1429 (2019).
  11. Giesel, F. L., et al. FAPI-74 PET/CT using either 18F-AlF or cold-kit 68Ga labeling: biodistribution, radiation dosimetry, and tumor delineation in lung cancer patients. J Nucl Med. 62 (2), 201-207 (2021).
  12. Jansen, K., et al. Selective inhibitors of fibroblast activation protein (FAP) with a (4-quinolinoyl)-glycyl-2-cyanopyrrolidine scaffold. ACS Med Chem Lett. 4 (5), 491-496 (2013).
  13. Tsai, T. -. Y., et al. Substituted 4-carboxymethylpyroglutamic acid diamides as potent and selective inhibitors of fibroblast activation protein. J Med Chem. 53 (18), 6572-6583 (2010).
  14. Zboralski, D., et al. Preclinical evaluation of FAP-2286 for fibroblast activation protein targeted radionuclide imaging and therapy. Eur J Nucl Med Mol Imaging. 49 (11), 3651-3667 (2022).
  15. Greifenstein, L., et al. 3BP-3940, a highly potent FAP-targeting peptide for theranostics - production, validation and first in human experience with Ga-68 and Lu-177. iScience. 26 (12), 108541 (2023).
  16. Kratochwil, C., et al. 68Ga-FAPI PET/CT: tracer uptake in 28 different kinds of cancer. J Nucl Med. 60 (6), 801-805 (2019).
  17. Guglielmo, P., et al. Head-to-head comparison of FDG and radiolabeled FAPI PET A systematic review of the literature. Life. 13 (9), 1821 (2023).
  18. Chen, H., et al. Usefulness of [68Ga]Ga-DOTA-FAPI-04 PET/CT in patients presenting with inconclusive [18F]FDG PET/CT findings. Eur J Nucl Med Mol Imaging. 48 (1), 73-86 (2021).
  19. Hicks, R. J., et al. FAPI PET/CT: Will it end the hegemony of 18F-FDG in oncology. JNucl Med. 62 (3), 296-302 (2021).
  20. Kuwert, T., et al. FAPI PET opens a new window to understanding immune-mediated inflammatory diseases. JNucl Med. 63 (8), 1136-1137 (2022).
  21. FAP inhibitor. (International publication no. WO 2019/154886 A1). WIPO Available from: https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019154886 (2019)
  22. SOFIE and GE HealthCare Enter Licensing Agreement to Develop FAP PET Radiotracers. GE Healthcare Available from: https://www.gehealthcare.com/about/newsroom/press-releases/sofie-and-ge-healthcare-enter-licensing-agreement-to-develop-fap-pet-radiotracers (2023)
  23. Meyer, C., et al. Radiation dosimetry and biodistribution of 68Ga-FAPI-46 PET imaging in cancer patients. JNucl Med. 61 (8), 1171-1177 (2020).
  24. Liu, Y., et al. Fibroblast activation protein targeted therapy using [177Lu]FAPI-46 compared with [225Ac]FAPI-46 in a pancreatic cancer model. Eur J Nucl Med Mol Imaging. 49 (3), 871-880 (2022).
  25. Meisenheimer, M., et al. Manual vs automated 68Ga-radiolabelling-A comparison of optimized processes. J Labelled Comp Radiopharm. 63, 162-173 (2020).
  26. Spreckelmeyer, S., et al. Fully automated production of [68Ga]Ga-FAPI-46 for clinical application. EJNMMI Radiopharm Chem. 5 (31), (2020).
  27. Boonkawin, N., Chotipanich, C. The first radiolabeled 68Ga-FAPI-46 for clinical PET applications using a fully automated iQS-TS synthesis system in Thailand. J Chulabhorn Royal Acad. 3 (3), 180-188 (2021).
  28. Nader, M., et al. [68Ga]/[90Y]FAPI-46: Automated production and analytical validation of a theranostic pair. Nucl Med Biol. 110-111, 37-44 (2022).
  29. Da Pieve, C., et al. New fully automated preparation of high apparent molar activity 68Ga-FAPI-46 on a Trasis AiO platform. Molecules. 27 (3), 675 (2022).
  30. Alfteimi, A., et al. Automated synthesis of [68Ga]Ga-FAPI-46 without pre-purification of the generator eluate on three common synthesis modules and two generator types. EJNMMI Radiopharm Chem. 7 (1), 20 (2022).
  31. Plhak, E., et al. Automated synthesis of [68Ga]Ga-FAPI-46 on a Scintomics GRP synthesizer. Pharmaceuticals. 16 (8), 1138 (2023).
  32. Rubira, L., et al. 68Ga]Ga-FAPI-46 synthesis on a GAIA® module system: Thorough study of the automated radiolabeling reaction conditions. Appl Radiat Isot. 206, 111211 (2024).
  33. Mallapura, , et al. Microfuidic-based production of [68Ga]Ga-FAPI-46 and [68Ga]Ga-DOTA-TOC using the cassette-based iMiDEV microfluidic radiosynthesizer. EJNMMI Radiopharm Chem. 8 (42), (2023).
  34. Rosenberg, A. J., Cheung, Y. -. Y., Sollert, C., Peterson, T. E., Kropski, J. A. Fully automated radiosynthesis of [68Ga] Ga-FAPI-46 with cyclotron produced gallium. EJNMMI Radiopharm Chem. 8 (29), (2023).
  35. Eryilmaz, K., Kilbas, B. Fully automated synthesis of 177Lu labelled FAPI derivatives on the module modular lab-Eazy. EJNMMI Radiopharm Chem. 6, 16 (2021).
  36. Greifenstein, L., et al. From automated synthesis to in vivo application in multiple types of cancer-clinical results with [68Ga]Ga-DATA5m.SA.FAPi. Pharmaceuticals. 15 (8), 1000 (2022).
  37. Haskali, M. B., et al. Automated preparation of clinical grade [68Ga]Ga-DOTA-CP04, a cholecystokinin-2 receptor agonist, using iPHASE MultiSyn synthesis platform. EJNMMI Radiopharm Chem. 4 (1), 23 (2019).
  38. Wichmann, C. W., et al. Automated radiosynthesis of [68Ga]Ga-PSMA-11 and [177Lu]Lu-PSMA-617 on the iPHASE MultiSyn module for clinical applications. J Labelled Comp Radiopharm. 64 (3), 140-146 (2021).
  39. Wichmann, C. W., et al. Automated radiosynthesis of [89Zr]Zr-DFOSq-Durvalumab for imaging of PD-L1 expressing tumours in vivo. Nucl Med Biol. , 120-121 (2023).
  40. European Pharmacopoeia. . European Pharmacopoeia. 11 edn. , (2024).
  41. Mu, L., et al. Identification, characterization and suppression of side-products formed during the synthesis of high dose 68Ga-DOTA-TATE. Appl Radiat Isot. 76, 63-69 (2013).

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Explore More Articles

Automated Radiosynthesis68Ga Ga FAPI 46Fibroblast Activation ProteinPositron Emission TomographyImaging TracerCancer ImagingPharmacokinetic PropertiesTumor RetentionFDG PETSolid Phase ExtractionClinical ApplicationsProduction OptimizationQuality Control Specifications

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2025 MyJoVE Corporation. All rights reserved