Automated Synthesis and Uptake Analysis of D-[Methyl-11C]-Methionine

Summary

Here, we present a protocol for the synthesis of D-[methyl-¹¹C]-methionine, a metabolic positron emission tomography tracer for bacteria, and in vitro evaluation in Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The synthesis involves a single synthetic step on an automated module, followed by microcentrifuge filtration.

Abstract

Positron emission tomography (PET) has emerged as a vital molecular imaging modality providing metabolic insights that are essential for the diagnosis and treatment of disease. Among the many useful applications of PET, imaging of infection has gained much traction in the last decade. To this end, we describe the synthesis and in vitro evaluation of D-[methyl-¹¹C]-methionine, a potent metabolic tracer for living bacteria. D-[methyl-¹¹C]-methionine was synthesized from [¹¹C]methyl iodide ([¹¹C]CH₃I) using an in-loop synthesis method in an automated synthesis module. The radiotracer was analyzed by chiral high-performance liquid chromatography (HPLC) to determine its radiochemical identity and purity and then subjected to in vitro analysis using a rapid method to determine uptake in bacteria cells. The workflow described demonstrates the importance of communication and time management when developing new radiotracers, especially with short half-life isotopes such as carbon-11 (¹¹C) (t_1/2 = 20.4 min).

Introduction

The use of new imaging techniques to detect infection is an emerging field and in recent years, PET imaging has been a leading innovator with several tracers specifically sensing living bacteria^{1,2,3,4,5,6,7,8}. D-[methyl-¹¹C]-methionine is a first-generation, metabolic PET tracer that images live bacterial infection by targeting peptidoglycan, a key cell wall component in both gram-positive and gram-negative bacteria pathogens⁹. Carbon-11 labelling of D-homocysteine easily affords the PET tracer in a single step from [¹¹C]CH₃I (Figure 1). [¹¹C]CH₃I is synthesized from [¹¹C]-carbon dioxide ([¹¹C]CO₂), which is cyclotron-generated. The synthetic process involves [¹¹C]CO₂ being reduced to [¹¹C]methane ([¹¹C]CH₄), which is subsequently reacted with iodine to yield [¹¹C]CH₃I.

The above process takes approximately 11 min, which is important because ¹¹C only has a half-life of 20.4 min. In general, for PET studies to yield enough radiotracer for in vitro studies, the radiosynthesis must not take any longer than 2–3 half-lives (40–60 min for ¹¹C) of the radioisotope¹⁰. As D-[methyl-¹¹C]-methionine is actively metabolized, high bacterial accumulation is anticipated due to enzymatic turnover compared to receptor-binding tracers. One major drawback of the use of the cyclic precursor (D-homocysteine thiolactone-HCl) is base-catalyzed, undesired epimerization resulting in the formation of the L-enantiomer of [methyl-¹¹C]-methionine.

Much like radiosynthesis, rapid pharmacokinetics is essential as it is important that localization of the tracer to regions of interest (cells, tissues) be observed quickly. For ¹¹C, imaging beyond 1–2 h is not feasible due to signal loss from decay. Therefore, quick internalization and retention is required for metabolic tracers like D-[methyl-¹¹C]-methionine. Any additional experiments will also require rapid processing for in vitro data collection to help identify tracers that will be successful in vivo.

Here, we describe the automated in-loop synthesis of D-[methyl-¹¹C]-methionine using ¹¹C methylation and the rapid analytical technique used for in vitro assessment of bacterial accumulation. The goal of this work is to provide an overview of the efficient methods needed to synthesize and analyze a ¹¹C radiotracer for PET molecular imaging studies. First, we review a plan to ensure the expedited chain of custody for the radioactivity as it navigates the synthesis, qualitative analysis, and in vitro experiments. Second, we describe a stepwise walkthrough of the preparation of the radiotracer and subsequent quality control (QC). Third, a workflow is provided for a rapid in vitro assay for the determination of bacterial uptake.

Protocol

CAUTION: In the following protocol, there are multiple manipulations that require the handling of radioactivity. It is extremely important that every interaction with radioactivity be executed in agreement with the Radiation Safety Department of the institute and the respective national guidelines. It is mandatory to minimize the exposure to ionizing radiation for the operators involved following the “as low as reasonably achievable” (ALARA) principle.

NOTE: “Current Good Manufacturing Procedures” (cGMP) are written into this protocol given that the synthesis and QC are done in the Clinical Production lab. cGMP is not required for preclinical use of this product or any other product. Requirements differ from facility to facility and these differences will be highlighted. Each synthesizer has module-specific programs to generate the [¹¹C]methyl iodide as well as product-specific programs for tracers. Herein, the “DMet Loop” program was modified using the program provided by the vendor and will need to be developed according to the facility’s capabilities.

1. Time management and planning of the experiment

NOTE: The ¹¹C nuclide has a short half-life of 20.4 min, making time management extremely important for all subsequent experiments to minimize loss of radioactivity (Figure 2). Any experiemental plan should ensure that specific personnel are responsible for different aspects of each experimental procedure, and coordinate between them with respect to timing and execution. For this experiment, four personnel are necessary: one for synthesis, one for QC, and two for preparation of the radionuclide and bacteria for the uptake assay and for performing the assay. To plan the experiment:

1. Organize time for the synthesis of the radiopharmaceutical based on the availability of isotope production and the synthesizer.
2. Inform the QC operator of the release criteria and specifications for preclinical use.
3. Ensure all components of the uptake assay are prepared.
4. Ensure a well-established chain of custody for the radiopharmaceutical from synthesis to experimental completion.

2. Automated synthesis of D-[methyl-¹¹C]-methionine for preclinical use

NOTE: Prepare the final product vial (FPV)-cGMP requirement at this facility.

1. Clean the laminar flow hood in accordance with cGMP standards, using standard operating procedures within the radiopharmaceutical facility.
2. Label air settling and contact plates for monitoring sterility.
3. Spray all components with sterile isopropyl alcohol (IPA), and place in the laminar flow hood.
4. Prepare a 20 mL evacuated vial with a sterile alcohol pad, vent needle, and 0.22 µm filter connected to an 18 G needle.
5. Perform environmental monitoring.
6. Dissolve 0.2 g of sodium dihydrogen phosphate in 1 mL of water to make a 0.2 g/mL solution. Add 1 mL of ethyl alcohol (EtOH) to a vial, then add 1 mL of 1 M NaOH to that vial, and label the vial as “50/50 0.5 M NaOH in water/EtOH”.
7. Synthesis module preparations
   1. Clean the synthesis module in accordance with the standard operating procedure (SOP) using ultra-high purity (UHP) water, sterile water for injection (SWFI), acetone, and EtOH; empty the vacuum trap, and clean the 2 mL Teflon loop with 5 mL water, 2 × 5 mL acetone, followed by drying with nitrogen gas flow of 30 mL/ min for 10 min. Verify that the hydrogen and helium gas tanks are open.
   2. Perform a manual leak check of the system prior to synthesis (Figure 3).
      1. Replace the large round bottom flask with a 10 mL dilution reservoir and small stir bar. Adjust tube lengths accordingly to ensure that the tube reaches the bottom of the 10 mL vial and that the vial is standing vertically.
      2. Remove the outlet of V7, and attach it to V14 in the dilution vessel.
      3. Initiate the manual system software, and leak check the system with the 2 mL loop placed between the reactor and V8 (Figure 3).
   3. Perform a final product line sanitization-cGMP if delivering to a dispensing cell.
      1. Attach the outlet of V13 to the final product delivery line in the dispensing cell.
      2. Place 5 mL of SWFI in reservoirs 5 and 6.
      3. Add SWFI into the product intermediate vial through manual manipulations of the software.
      4. Transfer the contents of the product intermediate vial through the delivery line into the dispensing cell and to the waste vial. Confirm the contents are in fact transferred to the dispensing cell and into the waste vial.
      5. Add 5 mL of EtOH to reservoirs 5 and 6. Repeat transfers in steps 2.7.3.3 and 2.7.3.4 through the product intermediate vial and to the dispensing cell.
      6. Add 10 mL of SWFI in reservoir 5 and repeat the transfers in steps 2.7.3.3 and 2.7.3.4. After the lines are clear of liquid, continue to purge with gas for 5 min to dry the delivery line.
      7. Close all valves on the synthesizer, exit the manual mode, and reset the system.
   4. Fill the cooling dewar with liquid nitrogen, and tighten the fittings.
   5. Add liquid nitrogen to the vacuum trap dewar (previously emptied during the cleaning of the module).
   6. Place 3 mL of saline in reservoir 2.
   7. Place 3 mL of saline in reservoir 3.
   8. Place 2 mL of saline in the product intermediate vial.
   9. Place 300 μL of 0.2 g/mL NaH₂PO₄ solution in the product intermediate vial.
   10. Activate a solid phase extraction (SPE) C18 light cartridge with 5 mL of EtOH, then 10 mL SWFI, and place in the SPE position (Figure 3).
   11. Ensure V7 to the right of V14 is attached such that V2 and V3 rinses pass into the dilution vial.
   12. Ensure that the round bottom flask has been replaced with a 10 mL dilution reservoir.
   13. Attach V12 to the overflow vial to collect the waste from loading the SPE cartridge.
   14. Attach the final product line to the dispensing cell delivery line.
   15. Ensure that the MeI trap is inserted into the heater (Figure 3).
8. Precursor preparation
   1. Remove the precursor vial from the refrigerator and allow it to reach room temperature (at least 20 min before dissolution).
   2. Dissolve the precursor in “50/50 0.5 M NaOH in water/EtOH” such that the final concentration is 1.25 mg in 100 μL.
   3. Using a 1 mL syringe with 25 G × 5/8” needle, withdraw 100 μL from the vial, and slowly add to the 2 mL Teflon loop, followed by 200 μL of air.
   4. Adjust the loop between the reaction vessel and V8 such that the side where the precursor was added from is toward V8 to allow the solution to spread into the loop before reaching the reactor when [¹¹C]methyl iodide is sent to the reactor.
9. Synthesis
   1. Start the “DMet Loop” program.
      1. Exit the manual mode and reset the software.
      2. Enter batch number (yymmddDMETx), where x is the sequential batch number of the day.
      3. Depending on the desired activity, start the program such that trap conditioning is done 20–30 min before the end of bombardment (EOB).
      4. Confirm that the actions in the following preparation list are performed.
         V1: Empty
         V2: 3 mL of saline are added.
         V3: 3 mL of saline are added.
         NaH₂PO₄ solution (0.2 g/mL, 300 μL) is added into the product intermediate vial
         Saline (2 mL) is added into the product intermediate vial.
         SPE C18 cartridge is conditioned and placed in the module.
         Precursor, as prepared above, is loaded onto the loop and attached between V8 and the reaction vessel.
10. Cyclotron-produced [¹¹C]CO₂
    1. Ensure that the magnet is turned on, and that the vacuum is appropriate. Empty and refill the carbon target.
    2. Choose the appropriate target and start the production with a current of 50–55 μA. When ready, select Start Irradiation, and wait for beam-on. Note that this production needs to be done concurrently with the presynthesis (module) preparation as the bombardment takes 20–40 min.
    3. When the desired activity is reached, and the synthesizer is ready to receive activity, select Delivery, and document the EOB.
    4. MeI production
       1. Condition the nickel oven until the beam is on target.
       2. Start cooling the methane trap 5 min before receiving [¹¹C]CO₂.
       3. Document the EOB time when starting the transfer of activity.
       4. Continue after the [¹¹C]CO₂ transfer is complete.
       5. Continue the synthesis continues according to the time list.
          NOTE: Following the delivery of [¹¹C]CO₂ from the cyclotron, the [¹¹C]CO₂ from the cyclotron is trapped on a column of molecular sieves and nickel at room temperature. The column is flushed with H₂ gas, sealed to pressurize, and finally heated to 350 °C to reduce [¹¹C]CO₂ to [¹¹C]CH₄. [¹¹C]CH₄ is then transferred into a methane trap that is previously cooled to -80 °C with liquid nitrogen. The methane trap is warmed to release [¹¹C]CH₄, which enters a recirculation loop containing an iodine reservoir at 90 °C at the entry point of the MeI oven set to 750 °C. The [¹¹C]CH₄ is converted to [¹¹C]CH₃I over a period of 5 min, and the converted [¹¹C]CH₃I is removed from the recirculation loop by a column that traps [¹¹C]CH₃I at room temperature, but allows [¹¹C]CH₄ to pass through. The [¹¹C]CH₃I is released upon heating of the column to 180 °C. The [¹¹C]CH₃I is flowed through the 2 mL Teflon loop, previously coated with the precursor for 70–90 s at a rate of 15 mL/min. The system is then closed to react for 1 min at room temperature. The loop is rinsed with 2 additions of 3 mL of saline into the 10 mL dilution flask, which is passed through the SPE C18 cartridge into the intermediate product vial previously charged with 300 μL of 0.2 g/mL NaH₂PO₄ and 2 mL of saline. This is transferred over a sterilizing filter to the final product vial.
          1. Note the activity on the detectors or readouts from the cyclotron: [¹¹C]CO₂ from the cyclotron, MeI counts, shine in the intermediate product vial, and final product activity.
    5. Post-synthesis
       1. Clean up the synthesizer following sufficient decay or on the day after removing the loop and placing all the tubing in the original position.
       2. Print rad detector reports and synthesis/ log reports.
11. Post-delivery
    1. After the product has been delivered, clear the filter of liquid. Remove the delivery line from the top of the sterilizing filter.
    2. Weigh the final vial and note the weight in the batch record.
    3. Assay the final vial in a dose calibrator. Calculate the concentration (mCi/mL).
    4. Remove the vial from the dose calibrator and take a QC sample (~0.6 mL).
    5. Inoculate sterility tubes (cGMP requirement for human doses, but not done for preclinical studies).
    6. Remove the sterilizing filter and vent needle. Put the final product vial back in the dose calibrator, and start half-life analysis (cGMP requirement, but not required for preclinical studies).

3. Quality control

1. Perform a visual inspection: clear, colorless, no particulate matter.
2. Perform a filter integrity test (bubble point): ≥50 psi (cGMP studies only).
3. Check the radiochemical purity (HPLC), that the radioactive peak corresponds to the standard peak.
4. Check the radiochemical purity (HPLC): ≥90%.
5. Check the radionuclidic identity (half-life): Run for at least 10 min; t_1/2 = 19.5–21 min (cGMP studies only).
6. Check the radionuclidic purity (MCA): 511 keV peak (cGMP studies only).
7. Check the pH: 4.5–7.5.
8. Check for bacterial endotoxins: <5 EU/mL (cGMP studies only).
9. Check for residual solvents (gas chromatography): EtOH ≤10% (cGMP studies only).
10. Start the sterility test: Started within 24 h (cGMP studies only).

4. Uptake assay

NOTE: All handling and experiments containing live bacteria (Figure 5) need to be within a designated biosafety cabinet within a biosafety level 2 (BSL-2) laboratory and in accordance with all safe practices and regulations at institutional, state, and federal levels. The majority of pathogenic bacteria are BSL-2.

1. Bacterial culture
   1. Remove a single colony of bacteria from an agar plate using a disposable loop and suspend in 50 mL of Luria-Bertani (LB) broth in a 125 mL culture flask.
   2. Clamp the flask in an incubator shaker and agitate at 111 rpm and at 37 °C for 16 h.
2. Prepare the bacteria for experiments.
   1. Remove an aliquot of 10 mL, and pellet at 1300 × g for 5 min.
   2. Remove the supernatant.
   3. Resuspend the pellet in 10 mL of Ham’s F12 medium.
   4. Aliquot the resuspended pellet into 50 mL centrifuge tubes containing a ½ dilution series of “cold” D-methionine ranging from 1 mM to 15.625 µM and 0 added D-methionine. Three experiments (centrifuge tubes) for each concentration are used for a total of 24 experiments.
3. Prepare the radiotracer for experiments.
   1. In a syringe, remove 3 mCi of the radiopharmaceutical, and dilute up to 1 mL in Ham’s F12 medium in a 2 mL centrifuge tube.
   2. Using a pipet, dispense 33.3 µL of activity to each bacterium-containing 50 mL centrifuge tube.
   3. Cap and seal each 50 mL centrifuge tube.
4. Perform the uptake assay.
   1. Place the rack of twenty-four 50 mL centrifuge tubes in an incubator shaker for agitation at 180 rpm for 90 min.
   2. After 90 min, remove 500 µL aliquots from each centrifuge tube, and place into 1.5 mL filtration tubes (for partitioning and analysis) and 1 mL UV/Vis cuvette (for final optical density (OD) determination).
   3. Centrifuge the 1.5 mL filtration tubes at 7,500 × g for 5 min.
   4. Wash the collected pellets with 200 µL of phosphate-buffered saline, and centrifuge at 7,500 × g for 5 min.
   5. Separate the collected pellets from the filtrates and close all containers.
5. Data collection
   1. Analyze the pellets and filtrates individually on a gamma counter. The gamma counter measures a window of 480–558 keV for 30 s for each sample.
   2. Analyze the aliquoted cuvettes on a UV/Vis spectrophotometer set to a wavelength of 600 nm.
6. Data analysis and processing
   1. Use the gamma counter to measure the amount of activity in each pellet and filtrate sample.
   2. Use the UV/Vis spectrophotometer to provide the OD of each bacterial sample.
   3. Use the OD to obtain the concentration of colony-forming units (CFU/mL) of the bacteria where ~1 OD⁶⁰⁰ corresponds to 8 x10⁸ CFU/mL as predetermined from plating and counting of the serial dilution series of each pathogen.
   4. Express the uptake initially as a percentage of activity in the pellet. At time zero, each 500 µL aliquot contains ~4.69 µCi of activity. Multiplication of the percentage provides the µCi of the accumulated radiotracer.
   5. Convert the µCi to Becquerel (Bq) using the conversion factor of 37000 Bq/µCi. The final expression of the cellular uptake data is Bq/million cells obtained from the CFUs and the activity at time zero.
   6. Plot Bq/million cells against the concentration of added “cold” D-methionine (Figure 6).

Results

The automated in-loop radiosynthesis of D-[methyl-¹¹C]-methionine yielded >99% enantiomeric excess (ee, n = 9), 22% ± 13% decay-corrected radiochemical yield, and >90% radiochemical purity in all cases. The overall synthesis required 20 min to complete, including 15 min dedicated to the synthesis of [¹¹C]methyl iodide. Labeling of the D-homocysteine precursors was completed in only 2–3 min and required passage through a C18 Sep Pak for isolation and purification. A small aliquot (...

Discussion

The radiosynthesis of D-[methyl-¹¹C]-methionine was performed in a commercial synthesis module using an in-loop method that yielded superior yields and purities over previously reported conventional manual approaches (>99% ee, 22% ± 13% decay-corrected radiochemical yield versus 85% ee, 20% ± 1% decay corrected radiochemical yield)⁹. When making radiotracers from either [¹¹C]carbon dioxide or [¹¹C] methyl iodide, is it imperative that a completely clo...

Materials

Name	Company	Catalog Number	Comments
Materials and instruments for radiosynthesis of D-[11C]methionine
0.5 M NaOH	Sigma-Aldrich, St. Louis, MO, USA
C18 Sep Pak	Waters, Milford, MA, USA
D/L-Methionine	Sigma-Aldrich, St. Louis, MO, USA		Standards
D-Homocysteine	Sigma-Aldrich, St. Louis, MO, USA		Precursor
Ethanol	Sigma-Aldrich, St. Louis, MO, USA
GE Medical Systems PET Trace	GE Healthcare, Uppsala Sweden		Cyclotron
Iodine	Merck, Darmstadt, Germany
Nickel Catalyst	Shimadzu, Kyoto, Japan
Nitrogen gas + 1% Oxygen	AirGas, Radnor, PA, USA		Target Gas
Saline	Sigma-Aldrich, St. Louis, MO, USA
Sodium Hydrogen Phosphate	Sigma-Aldrich, St. Louis, MO, USA
TRACERlab FXC	GE Healthcare, Uppsala Sweden		Synthesis Module
Water	Sigma-Aldrich, St. Louis, MO, USA
Materials and instruments for quality control of D-[11C]methionine
Acetonitrile	Sigma-Aldrich, St. Louis, MO, USA
Agilent 8890	Agilent, Santa Clara, CA, USA		Analytical GC
Bioscan AR2000	Bioscan, Santa Barbara, CA, USA		RadioTLC
Chirobiotic T2 column (250 x 4.6 mm)	Astec, Chattanooga, TN, USA		Chiral HPLC Column
Water	Sigma-Aldrich, St. Louis, MO, USA
Waters 600 Controller	Waters, Milford, MA, USA		Analytical HPLC
Materials and instruments for bacterial uptake assays of D-[11C]methionine
15 mL Centrifuge Tubes	Corning, NY, USA
50 mL Bio-Reaction Tubes	Celltreat, MA, USA
E. Coli	ATCC, Manassa, VA, USA		25922
F12 Media	Thermo, Waltham, MA, USA
Genesys 20	Thermo, Waltham, MA, USA		UV/Vis Spectrometer
Hidex AMG	Hidex, Turku, Finland		Gamma Counter
Innova 42	Brunswick, Lake Forest, IL, USA		Incubator/Shaker
LB Agar Plates	Teknova, Hollister, CA, USA
LB Broth	Teknova, Hollister, CA, USA
P. Aeruginosa	ATCC, Manassa, VA, USA		10145
S. Aureus	ATCC, Manassa, VA, USA		12600
Spin-X Filter Tubes	Corning, NY, USA
UV Cuvettes	Fisher, Waltham, MA, USA