protocol
Optimization of nucleophilic 18F radiofluorinations using a microfluidic reaction approach Giancarlo Pascali1, Lidia Matesic1, Thomas L Collier2, Naomi Wyatt1, Benjamin H Fraser1, Tien Q Pham1, Piero A Salvadori3 & Ivan Greguric1 1LifeSciences Division, Australian Nuclear Science and Technology Organisation, Lucas Heights, New South Wales, Australia. 2Department of
Radiology, Massachusetts General Hospital, Boston, Massachusetts, USA. 3Institute of Clinical Physiology, National Research Council, Pisa, Italy. Correspondence should be addressed to G.P. ([email protected]).
© 2014 Nature America, Inc. All rights reserved.
Published online 31 July 2014; doi:10.1038/nprot.2014.137
Microfluidic techniques are increasingly being used to synthesize positron-emitting radiopharmaceuticals. Several reports demonstrate higher incorporation yields, with shorter reaction times and reduced amounts of reagents compared with traditional vessel-based techniques. Microfluidic techniques, therefore, have tremendous potential for allowing rapid and cost-effective optimization of new radiotracers. This protocol describes the implementation of a suitable microfluidic process to optimize classical 18F radiofluorination reactions by rationalizing the time and reagents used. Reaction optimization varies depending on the systems used, and it typically involves 5–10 experimental days of up to 4 h of sample collection and analysis. In particular, the protocol allows optimization of the key fluidic parameters in the first tier of experiments: reaction temperature, residence time and reagent ratio. Other parameters, such as solvent, activating agent and precursor concentration need to be stated before the experimental runs. Once the optimal set of parameters is found, repeatability and scalability are also tested in the second tier of experiments. This protocol allows the standardization of a microfluidic methodology that could be applied in any radiochemistry laboratory, in order to enable rapid and efficient radiosynthesis of new and existing [18F]-radiotracers. Here we show how this method can be applied to the radiofluorination optimization of [18F]-MEL050, a melanoma tumor imaging agent. This approach, if integrated into a good manufacturing practice (GMP) framework, could result in the reduction of materials and the time required to bring new radiotracers toward preclinical and clinical applications.
INTRODUCTION [18F]-Fluorinated radiopharmaceuticals are widely used in positron emission tomography (PET) for performing high-impact molecular imaging experiments1. The most common way to introduce the [18F]-fluoride nuclide is via a nucleophilic substitution reaction pathway using appropriate substrates2. When searching for new diagnostic leads, it is generally required to test different radiolabeling conditions or precursors, often based on different molecular scaffolds, to choose the best set of conditions leading to a practically useful amount of a radiopharmaceutical for PET imaging evaluation. Fast-tracking radiochemistry optimization processes could thus permit high-impact outcomes in molecular imaging. Microfluidic reactor technology not only can increase radiochemical yields but also can decrease reaction times, decrease the amounts of radiolabeling precursors and minimize the radiation burden to the operator. Microfluidic reactors are proving vastly superior to vessel reactors in optimizing radiolabeling reactions, owing to larger amounts of optimization data being obtained in markedly less time. As almost all radiolabeling precursors are purchased from commercial suppliers or synthesized via costly multistep reaction sequences, microfluidic reactors can substantially decrease the cost of developing new radiopharmaceuticals 3–5. For these reasons, this approach is increasingly being adopted by world-class radiochemistry facilities for synthesizing and developing PET radiopharmaceuticals6–11. Nevertheless, thus far there has been a wide variety of experiences, tests and priorities among laboratories, so that no homogenous optimization approach is currently available. By starting from some chemical assumptions regarding the nature of the precursor, solvent and activating agent, this protocol
would allow a researcher to rationalize the use of time, radio active starting material and precursor mass needed when facing the challenge of optimizing the radioactive incorporation of [18F]-fluoride into novel molecular structures. It involves the use of a microfluidic system with precise features (e.g., Box 1) and uses premade solutions of precursor and fluorination complex (e.g., Box 2) to assess the best flow parameters. Accordingly, we propose some criteria that can be used to choose the best set of parameters, and we place particular attention on the repeatability and scalability of this optimized process. 18F radiofluorination features
[ 18F]-Fluorine (109 min half-life) is often described as the ‘ideal’ PET radioisotope because of a combination of highly favorable physical and chemical characteristics. It can be easily produced as aqueous fluoride by means of proton irradiation of 18O-enriched water at a biomedical cyclotron, whereas electrophilic [18F]-fluorine is typically less available. The concentration of available fluoride anions is generally well below the micromolar range 12, and therefore its use in radiolabeling processes needs to account for this small concentration and for the short half-life. The 511-keV radiation from the positronelectron annihilation requires all handling to be conducted according to radiation-protection rules and, in general, behind adequate shielding barriers; the use of automated or remotely controlled equipment is therefore frequent and necessary to achieve a substantial reduction of operator exposure. Several technical solutions have been proposed as radiochemical platforms, and their implementation directly affects radiolabeling performances and processes throughput. Recent reviews have nature protocols | VOL.9 NO.9 | 2014 | 2017
protocol Box 1 | Advion NanoTek microfluidic system for optimization Reaction i Flow iP3 P3 Volume iP3
Sweep i Flow iP3 P3 Volume iP3 Ti
Flow iP1 Volume iP1
P1
P1
© 2014 Nature America, Inc. All rights reserved.
The setup provided by Advion (http://www.advion.com/products/nanotek/) allows the fast implementation of several back-to-back reactions, by using two bridged 8-position valves (P1 and P3) each connected to a precision metering syringe and operating in the software ‘Discovery Mode’. In the first phase of each i-th run, reaction solvent in the syringe is used to deliver the desired volume of reagents at a set flow rate from the dedicated storage loops (in orange) into the microreactor, heated at the required temperature Ti. In the second phase, only one of the two pumps is used to clean (‘sweep’) the reactor with the minimum amount of solvent required to clean all the tubing up to the collection vial. In this apparatus, the reactor is a coil of fused silica with an internal volume of 15.6 or 31.2 µl, interfaced with three inlets and one outlet.
been dedicated to both conventional (vessel chemistry) and microfluidic platforms13. The most frequently used route of 18F radiolabeling is based on the use of nucleophilic substitution reactions on suitable precursors carrying appropriate leaving groups (e.g., halides, sulfonates and ammonium cations), and in the case of an aromatic ring, the concomitant presence of residues activating such reactions (e.g., electron-withdrawing groups in appropriate positions)14. In addition, owing to the strong hydration energy of fluoride in water that considerably reduces the nucleophilic character of the anion, the crude [18F]-fluoride produced from the cyclotron generally needs to be dried and solvated in organic polar aprotic solvents15, although there are a few procedures that have been recently reported that do not require this drying step16–18. To increase the nucleophilicity of fluoride in these organic solvents, activating agents such as quaternary ammonium salts or inorganic
bases (e.g., K2CO3, KHCO3 and Cs2CO3) with or without phasetransfer catalysts need to be added to the mixture. In the classical vessel chemistry, the 18F radiofluorination reaction is generally performed at medium-to-high temperatures (>90 °C) for short periods of time (5–30 min) and in small volumes (
Optimization of nucleophilic 18F radiofluorinations using a microfluidic reaction approach Giancarlo Pascali1, Lidia Matesic1, Thomas L Collier2, Naomi Wyatt1, Benjamin H Fraser1, Tien Q Pham1, Piero A Salvadori3 & Ivan Greguric1 1LifeSciences Division, Australian Nuclear Science and Technology Organisation, Lucas Heights, New South Wales, Australia. 2Department of
Radiology, Massachusetts General Hospital, Boston, Massachusetts, USA. 3Institute of Clinical Physiology, National Research Council, Pisa, Italy. Correspondence should be addressed to G.P. ([email protected]).
© 2014 Nature America, Inc. All rights reserved.
Published online 31 July 2014; doi:10.1038/nprot.2014.137
Microfluidic techniques are increasingly being used to synthesize positron-emitting radiopharmaceuticals. Several reports demonstrate higher incorporation yields, with shorter reaction times and reduced amounts of reagents compared with traditional vessel-based techniques. Microfluidic techniques, therefore, have tremendous potential for allowing rapid and cost-effective optimization of new radiotracers. This protocol describes the implementation of a suitable microfluidic process to optimize classical 18F radiofluorination reactions by rationalizing the time and reagents used. Reaction optimization varies depending on the systems used, and it typically involves 5–10 experimental days of up to 4 h of sample collection and analysis. In particular, the protocol allows optimization of the key fluidic parameters in the first tier of experiments: reaction temperature, residence time and reagent ratio. Other parameters, such as solvent, activating agent and precursor concentration need to be stated before the experimental runs. Once the optimal set of parameters is found, repeatability and scalability are also tested in the second tier of experiments. This protocol allows the standardization of a microfluidic methodology that could be applied in any radiochemistry laboratory, in order to enable rapid and efficient radiosynthesis of new and existing [18F]-radiotracers. Here we show how this method can be applied to the radiofluorination optimization of [18F]-MEL050, a melanoma tumor imaging agent. This approach, if integrated into a good manufacturing practice (GMP) framework, could result in the reduction of materials and the time required to bring new radiotracers toward preclinical and clinical applications.
INTRODUCTION [18F]-Fluorinated radiopharmaceuticals are widely used in positron emission tomography (PET) for performing high-impact molecular imaging experiments1. The most common way to introduce the [18F]-fluoride nuclide is via a nucleophilic substitution reaction pathway using appropriate substrates2. When searching for new diagnostic leads, it is generally required to test different radiolabeling conditions or precursors, often based on different molecular scaffolds, to choose the best set of conditions leading to a practically useful amount of a radiopharmaceutical for PET imaging evaluation. Fast-tracking radiochemistry optimization processes could thus permit high-impact outcomes in molecular imaging. Microfluidic reactor technology not only can increase radiochemical yields but also can decrease reaction times, decrease the amounts of radiolabeling precursors and minimize the radiation burden to the operator. Microfluidic reactors are proving vastly superior to vessel reactors in optimizing radiolabeling reactions, owing to larger amounts of optimization data being obtained in markedly less time. As almost all radiolabeling precursors are purchased from commercial suppliers or synthesized via costly multistep reaction sequences, microfluidic reactors can substantially decrease the cost of developing new radiopharmaceuticals 3–5. For these reasons, this approach is increasingly being adopted by world-class radiochemistry facilities for synthesizing and developing PET radiopharmaceuticals6–11. Nevertheless, thus far there has been a wide variety of experiences, tests and priorities among laboratories, so that no homogenous optimization approach is currently available. By starting from some chemical assumptions regarding the nature of the precursor, solvent and activating agent, this protocol
would allow a researcher to rationalize the use of time, radio active starting material and precursor mass needed when facing the challenge of optimizing the radioactive incorporation of [18F]-fluoride into novel molecular structures. It involves the use of a microfluidic system with precise features (e.g., Box 1) and uses premade solutions of precursor and fluorination complex (e.g., Box 2) to assess the best flow parameters. Accordingly, we propose some criteria that can be used to choose the best set of parameters, and we place particular attention on the repeatability and scalability of this optimized process. 18F radiofluorination features
[ 18F]-Fluorine (109 min half-life) is often described as the ‘ideal’ PET radioisotope because of a combination of highly favorable physical and chemical characteristics. It can be easily produced as aqueous fluoride by means of proton irradiation of 18O-enriched water at a biomedical cyclotron, whereas electrophilic [18F]-fluorine is typically less available. The concentration of available fluoride anions is generally well below the micromolar range 12, and therefore its use in radiolabeling processes needs to account for this small concentration and for the short half-life. The 511-keV radiation from the positronelectron annihilation requires all handling to be conducted according to radiation-protection rules and, in general, behind adequate shielding barriers; the use of automated or remotely controlled equipment is therefore frequent and necessary to achieve a substantial reduction of operator exposure. Several technical solutions have been proposed as radiochemical platforms, and their implementation directly affects radiolabeling performances and processes throughput. Recent reviews have nature protocols | VOL.9 NO.9 | 2014 | 2017
protocol Box 1 | Advion NanoTek microfluidic system for optimization Reaction i Flow iP3 P3 Volume iP3
Sweep i Flow iP3 P3 Volume iP3 Ti
Flow iP1 Volume iP1
P1
P1
© 2014 Nature America, Inc. All rights reserved.
The setup provided by Advion (http://www.advion.com/products/nanotek/) allows the fast implementation of several back-to-back reactions, by using two bridged 8-position valves (P1 and P3) each connected to a precision metering syringe and operating in the software ‘Discovery Mode’. In the first phase of each i-th run, reaction solvent in the syringe is used to deliver the desired volume of reagents at a set flow rate from the dedicated storage loops (in orange) into the microreactor, heated at the required temperature Ti. In the second phase, only one of the two pumps is used to clean (‘sweep’) the reactor with the minimum amount of solvent required to clean all the tubing up to the collection vial. In this apparatus, the reactor is a coil of fused silica with an internal volume of 15.6 or 31.2 µl, interfaced with three inlets and one outlet.
been dedicated to both conventional (vessel chemistry) and microfluidic platforms13. The most frequently used route of 18F radiolabeling is based on the use of nucleophilic substitution reactions on suitable precursors carrying appropriate leaving groups (e.g., halides, sulfonates and ammonium cations), and in the case of an aromatic ring, the concomitant presence of residues activating such reactions (e.g., electron-withdrawing groups in appropriate positions)14. In addition, owing to the strong hydration energy of fluoride in water that considerably reduces the nucleophilic character of the anion, the crude [18F]-fluoride produced from the cyclotron generally needs to be dried and solvated in organic polar aprotic solvents15, although there are a few procedures that have been recently reported that do not require this drying step16–18. To increase the nucleophilicity of fluoride in these organic solvents, activating agents such as quaternary ammonium salts or inorganic
bases (e.g., K2CO3, KHCO3 and Cs2CO3) with or without phasetransfer catalysts need to be added to the mixture. In the classical vessel chemistry, the 18F radiofluorination reaction is generally performed at medium-to-high temperatures (>90 °C) for short periods of time (5–30 min) and in small volumes (
