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

Carbon-11 is one of the most widely used radioisotopes in positron emission tomography because of its abundance in organic molecules and a short half-life of 20 minutes. In this video, we show an efficient carbon-11 radiolabeling technique using solid-phase extraction cartridges. Compared to conventional methods, cartridge-based techniques obviates the use of HPLC, shortens the radiosynthesis time, improves synthesis reliability, simplifies automation process, and facilitates compliance with the Good Manufacturing Practices, GMP.

The cartridge-based technique is demonstrated here on the radiosynthesis of C11 PiB, a PET tracer used for the in vivo imaging of amyloid plaques in the brains of patients suffering from Alzheimer's disease. Recently, we applied the three-in-one technique to the radiosynthesis of C11 ABP688, a PET tracer for the imaging of metabotropic glutamate receptors type five, as well as other carbon-11 labeled tracers. Combine 450 milliliters of 0.2-molar solution of acetic acid with 50 milliliters of 0.2-molar solution of sodium acetate to prepare the acetate buffer at pH 3.7 as buffer one.

Verify the pH of the buffer with pH strips or a pH meter. Then, combine 12.5 milliliters of absolute ethanol with 87.5 milliliters of acetate buffer in a 100-milliliter bottle to make 12.5%aqueous ethanol solution as wash one. Combine 15 milliliters of absolute ethanol with 85 milliliters of acetate buffer in a 100-milliliter bottle to make 15%aqueous ethanol solution as wash two.

Combine five milliliters of absolute ethanol with five milliliters of acetate buffer to make 50%aqueous ethanol solution as final eluent, and draw 2.5 milliliters of this solution into a 10-milliliter syringe. To precondition the tC18 cartridge, from the female end, use a syringe to pass 10 milliliters of water followed by five milliliters of acetone through the cartridge. Dry the cartridge with a stream of nitrogen at 50 milliliters per minute for one minute.

In an Eppendorf tube, dissolve two milligrams of the precursor 6-OH-BTA-0 in one milliliter of anhydrous acetone. Holding a Luer-tip, 250-microliter, precision glass syringe downwards, withdraw 100 microliters of the precursor solution and then 50 microliters of air cushion. Remove the needle, and tap the syringe to make sure the air cushion is above the solution in a syringe.

Apply the precursor solution to the tC18 cartridge from the female end by slowly pushing the plunger all the way down. Do not push the air any further. Secure and assemble the standard five-port disposable manifold on the synthesis module.

Port one has two positions. Connect the horizontal inlet to the automated dispenser fitted with a 20-milliliter syringe. Connect the vertical inlet to the bottle with wash one.

Connect the output of the module which produces C11 methyl triflate to port two of the manifold. Install the tC18 cartridge loaded with precursor 6-OH-BTA-0 between ports three and four. Port five has two positions.

Connect the horizontal outlet to the waste bottle and the vertical outlet to the sterile vial for tracer collection via the sterile filter. In the lead-shielded hot cell, use a Teflon line to deliver C11 methyl triflate into the manifold through port two, and pass it through the loaded tC18 cartridge at 20 milliliters per minute output flow. The C11 methyl triflate module regulates the flow via ports three and four and into the waste bottle.

Once all the radioactivity has been transferred and trapped in the tC18 cartridge as monitored by the radioactivity detector, stop the flow of gas by closing port two. Let the cartridge sit for two minutes to complete the reaction. Then, through port one, withdraw 19 milliliters of wash one solution from the 100-milliliter bottle into the dispenser syringe at 100 milliliters per minute.

Dispense 18.5 milliliters of wash one solution from the dispenser through the tC18 cartridge via ports three and four and into the waste bottle at 50 milliliters per minute. Ensure the absence of air bubbles in the manifold, as they might diminish the separation efficiency. Repeat the withdrawing and dispensing four times with 18.5 milliliters of wash one solution each time and a total volume of 92.5 milliliters passing through tC18.

Switch the input line on port one from wash one to wash two. Repeat the withdrawing and dispensing three times with 18.5 milliliters of wash two solution each time and total volume of 55.5 milliliters passing through tC18. Toggle valve five towards the final vial.

Disconnect the line from the dispenser, and connect it to the 10-milliliter syringe containing 2.5 milliliters of the final eluent solution and 7.5 milliliters of air. Holding the syringe downwards, manually push the final eluent solution followed by the air through the tC18 cartridge via ports three and four and into the sterile vial for tracer collection via the sterile filter. Switch the syringe to the one containing 10 milliliters of the sterile phosphate buffer, and push the entire volume through the tC18 cartridge into the sterile vial.

Disconnect the syringe, and flush the line with 10 milliliters of air using the same syringe. Using one-milliliter syringe, withdraw samples for prerelease quality control procedures, bacterial endotoxin test, and sterility test. To perform prerelease quality control procedures, first determine the radiochemical identity, radiochemical purity, chemical purity, and molar activity of the tracer by an analytical HPLC system equipped with UV 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. Determine the residual solvent content by the 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.

This study performed the radiosynthesis of C11 PiB by C11-methylation of 6-OH-BTA-0 precursor with C11 methyl triflate. Quality control analytical HPLC of C11 PiB shows the radiochemical purity was 98%The retention times of 6-OH-BTA-0 precursor and 6-OH-BTA-1 tracer peak on the UV chromatogram were 3.6 and 5.9 minutes, respectively. The analysis of the UV trace shows residual precursor concentration below the acceptable limit of 1.3 micrograms in the absence of other non-radioactive impurities.

This indicates the radiochemical and chemical purity of the tracer is acceptable for clinical PET studies. Increasing the 6-OH-BTA-0 precursor amount from 0.1 milligrams to 0.3 milligrams improved the radiochemical yield from 18.1%to 32.1%at the expense of a slightly higher amount of the precursor in the final product. This technique should be applicable to many different systems with a suitable difference in polarity between the precursor and radiolabeled product.

All manipulations involving radioactive isotopes must be performed in a lead-shielded hot cell by personnel with adequate training to handle radioactive materials.