Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

Summary

We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. ¹¹C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields.

Abstract

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a non-radioactive precursor in solution. This approach necessitates purification of the tracer by high performance liquid chromatography (HPLC) followed by reformulation in a biocompatible solvent for human administration. We recently developed a novel ¹¹C-methylation approach for the highly efficient synthesis of carbon-11 labeled PET radiopharmaceuticals, taking advantage of solid phase cartridges as disposable "3-in-1" units for the synthesis, purification and reformulation of the tracers. This approach obviates the use of preparative HPLC and reduces the losses of the tracer in transfer lines and due to radioactive decay. Furthermore, the cartridge-based technique improves synthesis reliability, simplifies the automation process and facilitates compliance with the Good Manufacturing Practice (GMP) requirements. Here, we demonstrate this technique on the example of production of a PET tracer Pittsburgh compound B ([¹¹C]PiB), a gold standard in vivo imaging agent for amyloid plaques in the human brains.

Introduction

Positron emission tomography (PET) is a molecular imaging modality which relies on detecting the radioactive decay of an isotope attached to a biologically active molecule to enable the in vivo visualization of biochemical processes, signals and transformations. Carbon-11 (t_1/2 = 20.3 min) is one of the most commonly used radioisotopes in PET because of its abundance in organic molecules and short half-life which allows for multiple tracer administrations on the same day to the same human or animal subject and reduces the radiation burden on the patients. Many tracers labeled with this isotope are used in clinical studies and in basic health research for in vivo PET imaging of classical and emerging biologically relevant targets - [¹¹C]raclopride for D₂/D₃ receptors, [¹¹C]PiB for amyloid plaques, [¹¹C]PBR28 for translocator protein - to name just a few.

Carbon-11 labeled PET tracers are predominantly produced via ¹¹C-methylation of non-radioactive precursors containing -OH (alcohol, phenol and carboxylic acid), -NH (amine and amide) or -SH (thiol) groups. Briefly, the isotope is generated in the gas target of a cyclotron via a ¹⁴N(p,α)¹¹C nuclear reaction in the chemical form of [¹¹C]CO₂. The latter is then converted into [¹¹C]methyl iodide ([¹¹C]CH₃I) via either wet chemistry (reduction to [¹¹C]CH₃OH with LiAlH₄ followed by quenching with HI)¹ or dry chemistry (catalytic reduction to [¹¹C]CH₄ followed by radical iodination with molecular I₂)². [¹¹C]CH₃I can then be further converted to the more reactive ¹¹C-methyl triflate ([¹¹C]CH₃OTf) by passing it over a silver triflate column³. The ¹¹C-methylation is then performed by either bubbling the radioactive gas into a solution of non-radioactive precursor in organic solvent or via the more elegant captive solvent "loop" method^4,5. The ¹¹C-tracer is then purified by means of HPLC, reformulated in a biocompatible solvent, and passed through a sterile filter before being administered to human subjects. All of these manipulations must be fast and reliable given the short half-life of carbon-11. However, the use of an HPLC system significantly increases the losses of the tracer and production time, often necessitates the use of toxic solvents, complicates automation and occasionally leads to failed syntheses. Furthermore, the required cleaning of the reactors and HPLC column prolongs delays between the syntheses of subsequent tracer batches and increases the exposure of personnel to radiation.

The radiochemistry of fluorine-18 (t_1/2 = 109.7 min), the other widely used PET isotope, has been recently advanced via the development of cassette-based kits that obviate the need for HPLC purification. By employing solid phase extraction (SPE) cartridges, these fully disposable kits allow for the reliable routine production of ¹⁸F-tracers, including [¹⁸F]FDG, [¹⁸F]FMISO, [¹⁸F]FMC and others, with shorter synthesis times, reduced personnel involvement and minimal maintenance of the equipment. One of the reasons carbon-11 remains a less popular isotope in PET imaging is a lack of similar kits for the routine production of ¹¹C-tracers. Their development would significantly improve synthetic reliability, increase radiochemical yields and simplify automation and preventive maintenance of the production modules.

Currently available production kits take advantage of inexpensive, disposable, SPE cartridges instead of HPLC columns for the separation of the radiotracer from unreacted radioactive isotope, precursor and other radioactive and non-radioactive by-products. Ideally, the radiolabeling reaction also proceeds on the same cartridge; for example, the [¹⁸F]fluoromethylation of dimethylaminoethanol with gaseous [¹⁸F]CH₂BrF in the production of prostate cancer imaging PET tracer [¹⁸F]fluoromethylcholine occurs on a cation-exchange resin cartridge⁶. Although similar procedures for the radiolabeling of several ¹¹C-tracers on cartridges have been reported^7,8 and became especially powerful for the radiosynthesis of [¹¹C]choline⁹ and [¹¹C]methionine¹⁰, these examples remain limited to oncological PET tracers where the separation from the precursor is often not required. We recently reported the development of "[¹¹C]kits" for the production of [¹¹C]CH₃I¹¹ and subsequent ¹¹C-methylation, as well as solid phase-supported synthesis¹² in our endeavours to simplify the routine production of ¹¹C-tracers. Here, we wish to demonstrate our progress using the example of the solid phase supported radiosynthesis of [¹¹C]PiB, a radiotracer for Aβ imaging which revolutionized the field of Alzheimer's disease (AD) imaging when it was first developed in 2003 (Figure 1)^13,14. In this method, volatile [¹¹C]CH₃OTf (bp 100 °C) is passed over 6-OH-BTA-0 precursor deposited on the resin of a disposable cartridge. PET tracer [¹¹C]PiB is then separated from the precursor and radioactive impurities by elution from the cartridge with biocompatible aqueous ethanol. Further, we automated this method of [¹¹C]PiB radiosynthesis using a remotely operated radiochemistry synthesis module and disposable cassette kits. Specifically, we implemented this radiosynthesis on a 20-valve radiochemistry module, equipped with syringe drive (dispenser) which fits standard 20 mL disposable plastic syringe, gas flow controller, vacuum pump and gauge. Due to the simplicity of this method, we are confident that it can be modified to most commercially available automated synthesizers, either cassette-based or those equipped with stationary valves. This solid phase supported technique facilitates [¹¹C]PiB production compliant with Good Manufacturing Practice (GMP) regulations and improves synthesis reliability. The technique described here also reduces the amount of precursor required for radiosynthesis, uses only "green" biocompatible solvents and decreases the time between consecutive production batches.

Protocol

1. Preparation of buffers and eluents

1. Dissolve 2.72 g of sodium acetate trihydrate in 100 mL of water to prepare 0.2 M sodium acetate solution (solution A).
2. Dissolve 11.4 mL of glacial acetic acid in 1 L of water to prepare 0.2 M acetic acid solution (solution B).
3. Combine 50 mL of solution A with 450 mL of solution B to prepare the acetate buffer at pH 3.7 (buffer 1) according to the buffer reference center¹⁵. Verify the pH of the buffer with pH strips or a pH meter.
4. Combine 12.5 mL of absolute ethanol with 87.5 mL of buffer 1 to make 12.5% aqueous EtOH solution (wash 1) in a 100 mL bottle.
5. Combine 15 mL of absolute ethanol with 85 mL of buffer 1 to make 15% aqueous EtOH solution (wash 2) in a 100 mL bottle.
6. Combine 5 mL of absolute ethanol with 5 mL of buffer 1 to make 50% aqueous EtOH solution (final eluent) and draw 2.5 mL of this solution into a 10 mL syringe.

2. Application of the precursor to the cartridge

1. Pass 10 mL of water followed by 5 mL of acetone through the tC18 cartridge to precondition it.
2. Dry the cartridge with a stream of nitrogen at 50 mL/min for 1 min.
3. Dissolve 2 mg of the precursor 6-OH-BTA-0 in 1 mL of anhydrous acetone.
4. Holding a Luer-tip 250 µL precision glass syringe downwards, withdraw 100 µL of the precursor solution and 50 µL of air cushion on top of the liquid. Remove the needle and apply the precursor solution on the tC18 cartridge from the female end by slowly pushing the plunger all the way down. Do not push the solution any further!

3. Setting up the manifold for automated synthesis

1. Secure the standard 5-port disposable manifold on the synthesis module and assemble it according to the Figure 2 and steps 3.2 - 3.5 below.
   NOTE: We recommend using acetone-resistant manifolds (see Table of Materials).
2. Port 1 has two positions. Connect the horizontal inlet to the automated dispenser fitted with a 20 mL syringe. Connect the vertical inlet to the bottle with wash 1.
3. Connect the output of the module which produces [¹¹C]CH₃OTf to port 2 of the manifold.
4. Install the tC18 cartridge loaded with precursor 6-OH-BTA-0 between ports 3 and 4.
5. Port 5 has two positions. Connect the horizontal outlet to the waste bottle which must hold at least 200 mL. Connect the vertical outlet to the sterile vial for tracer collection via the sterile filter.

4. Radiosynthesis of [¹¹C]PiB

CAUTION: All manipulations involving radioactive isotopes must be performed in a lead-shielded hot cell by personnel with adequate training to work with radioactive materials.
NOTE: This protocol does not cover the details of production of [¹¹C]CO₂ in the cyclotron and its conversion into [¹¹C]CH₃OTf using the radiochemistry module. These procedures will depend on the individual equipment of the radiochemistry lab and are outside the scope of this protocol. Our PET centre is equipped with an IBA cyclotron, which produces carbon-11 in the chemical form of [¹¹C]CO₂ via the ¹⁴N(p,α)¹¹C nuclear reaction with a N₂/O₂ gas mixture (99.5:0.5) in the gas target, and a commercially available module for production of [¹¹C]CH₃I via the "dry method" (catalytic reduction to [¹¹C]CH₄ followed by radical iodination). [¹¹C]CH₃OTf is produced by passing [¹¹C]CH₃I over a silver triflate column heated to 175 °C at 20 mL/min.

1. Deliver [¹¹C]CH₃OTf into the manifold through port 2 and pass it through the loaded tC18 cartridge at 20 mL/min output flow regulated by the [¹¹C]CH₃OTf module, via ports 3 and 4 and into the waste bottle as shown on Figure 2A.
2. Once all the radioactivity has been transferred and trapped on the tC18 cartridge as monitored by the radioactivity detector behind the cartridge holder, stop the flow of gas by closing port 2. Let the cartridge sit for 2 min to complete the reaction.
3. Withdraw 19 mL of wash 1 solution (see step 1.4) from the 100 mL bottle into the dispenser syringe through port 1 at 100 mL/min as shown on Figure 2B.
4. Dispense 18.5 mL of wash 1 solution from the dispenser through the tC18 cartridge via ports 3 and 4 and into the waste bottle at 50 mL/min as shown on Figure 2C. Ensure the absence of air bubbles in the manifold as they might diminish the separation efficiency.
5. Repeat steps 4.3 and 4.4 four times, withdrawing and dispensing 18.5 mL of wash 1 solution each time. The total volume of wash 1 solution passed through tC18 is 92.5 mL; however, it can vary within the 90 - 100 mL range depending on the particular synthesis module used.
6. Switch the input line on port 1 from wash 1 to wash 2 solution (see step 1.5).
7. Repeat steps 4.3 and 4.4 three times, withdrawing and dispensing 18.5 mL of wash 2 solution each time. The total volume of wash 2 solution passed through tC18 is 55.5 mL. However, it can vary within the 50 - 60 mL range depending on the particular synthesis module used.
8. Toggle valve 5 towards the final vial as shown on Figure 2D. Disconnect the line from the dispenser and connect it to the 10 mL syringe containing 2.5 mL of the final eluent solution (50% aqueous EtOH, see step 1.6) and 7.5 mL of air.
9. Holding the syringe downwards, manually push the final eluent solution (2.5 mL) followed by air (7.5 mL) through the tC18 cartridge via ports 3 and 4 and into the sterile vial for tracer collection via the sterile filter as shown on Figure 2D.
10. Disconnect the empty syringe, connect the 10 mL syringe containing 10 mL of the sterile phosphate buffer (recipe not included as it may vary) and push the entire volume through the tC18 cartridge into the sterile vial as described above (Figure 2D). Disconnect the syringe and flush the line with 10 mL of air using the same syringe.
11. Withdraw 0.7 mL of the final tracer formulation and collect samples for quality control procedures (0.1 mL), bacterial endotoxin test (0.1 mL) and sterility (0.5 mL).

5. Quality control procedures

CAUTION: Each batch of the radiotracer must be subjected to the appropriate quality control procedures (QC) prior to release to the PET imaging site for administration into human or animal subjects. The authors of this manuscript are not responsible for the compliance of the radiotracer produced at other centers with local health authority regulations.

1. Perform pre-release QC procedures, which must include tests for radiochemical identity (RCI), radiochemical purity (RCP), chemical purity and molar activity of the tracer as well as residual solvent content and pH of the formulation.
2. Determine the RCI, RCP, chemical purity and molar activity by means of analytical HPLC system equipped with UV (monitoring at 350 nm) and radioactivity detectors, and a reversed-phase column. Determine the retention times of 6-OH-BTA-0 and 6-OH-BTA-1 and calibrate the instrument to quantify the content of each compound.
3. Determine the residual solvent content by means of analytical gas chromatography system equipped with a capillary column. Determine the retention times of acetone and ethanol and calibrate the instrument to quantify the content of each solvent.
4. Perform the bacterial endotoxins test using a cartridge reader equipped with suitable cartridges.
5. Perform the sterility analysis of the sample at least 14 day after the synthesis to ensure the absence of bacterial growth or send the sterility sample to a laboratory accredited by the local health authority.

Results

To summarize a typical radiosynthesis of [¹¹C]PiB, gaseous [¹¹C]CH₃OTf is first passed through a tC18 cartridge preloaded with a solution of precursor (Figure 1). Separation of the reaction mixture is then achieved by successive elution with aqueous ethanol solutions as follows. First, 12.5% EtOH elutes the majority of unreacted [¹¹C]CH₃OTf and 6-OH-BTA-0, then 15% EtOH washes out the residual impurities,...

Discussion

Despite the recent emergence and FDA approval of several ¹⁸F-labeled PET tracers, such as florbetapir, florbetaben and flutemetamol, [¹¹C]PiB remains a gold standard tracer for amyloid imaging due to the fast brain uptake and low non-specific binding. Currently this tracer is synthesized via either wet chemistry¹⁶ or using a "dry loop" approach^4,17. Both methods require HPLC purification followed by r...

Materials

Name	Company	Catalog Number	Comments
6-OH-BTA-0	ABX advanced biochemical compounds	5101	Non-radioactive precursor of [11C]PiB
6-OH-BTA-1	ABX advanced biochemical compounds	5140	Non-radioactive standard of [11C]PiB
Agilent 1200 HPLC system	Agilent	Agilent 1200	Analytical HPLC system
Ethanol absolute	Commercial alcohols	432526
Hamilton syringe (luer-tip, 250 µL)	Hamilton	HAM80701
MZ Analytical PerfectSil 120	MZ-Analysentechik GmbH	MZ1440-100040	Analytical HPLC column
Perkin Elmer Clarus 480 GC system	Perkin Elmer	Clarus 480	Gas chromotograph
polycarbonate manifold	Scintomics	ACC-101	Synthesis manifold
Restek MTX-Wax column (30 m, 0.53 mm)	Restek	70625-273	Analytical GC column
Scintomics GRP module	Scintomics	Scintomics GRP	Automated synthesis unit
Sep-Pak tC18 Plus	Waters	WAT020515	Solid phase extraction cartridge
solvent-resistant manifold	Scintomics	ACC-201	Synthesis manifold
Spinal needle	BD	405181
Sterile extension line	B. Braun	8255059
Sterile filter	Millipore	SLLG013SL
Sterile vial (20mL)	Huayi	SVV-20A
Sterile water	Baxter	JF7623
Synthra MeIplus Research	Synthra	MeIplus Research	[11C]CH3I/[11C]CH3OTf module
Syringe (10 mL)	BD	309604
Syringe (1mL)	BD	309659
Syringe (20 mL)	B. Braun	4617207V	Dispenser syringe
Vent filter	Millipore	TEFG02525