Opportunities and Challenges in the Utilization of Microfluidic Technologies to the Production of Radiopharmaceuticals

GIANCARLO PASCALI*, PIERO A. SALVADORI *Corresponding author 1. Australian Nuclear Science and Technology Organization, LifeSciences Institute1, Grose St, Camperdown 2050, NSW, Australia 2. Consiglio Nazionale delle Ricerche, Istituto di Fisiologia Clinica, Via Moruzzi 1, 56100 Pisa, Italy

Giancarlo Pascali

Opportunities and challenges in the utilization of microfluidic technologies to the production of radiopharmaceuticals

KEYWORDS: Microfluidics, radiopharmaceuticals, PET, nuclear medicine, flow chemistry, tracer, radiofluorination.

Microfluidics and flow chemistry techniques are gaining an increasing importance in the set-up of Abstract chemical production processes. Radiopharmaceutical chemistry is one of the most delicate fields of medicinal products’ production, in which microchemistry combines with the use of short-lived nuclides. In this perspective, we will show how microfluidic techniques, a subset of flow chemistry approaches, is changing the methodological scenario by introducing sensible improvements in the synthesis of radiopharmaceuticals, widely used in diagnosis and research. Opportunities, problems and challenges will be discussed, as well as new application in the radiochemistry field, which may translate to potential future developments for applied research.

INTRODUCTION industry (including microelectronic and engineering), microfl uidics is mutating from a replacement technology to Microﬂ uidics in chemical world an enabling one. In general, the best success of microfl uidics The development of a chemical process involves the full has been achieved whenever the process needed to be moved journey from selecting and combining the starting materials, closer to the end user, such as in the case of point-of-care or down to the fi nished product (Figure 1). This task has to lab-on-chips for environmental or medical control. respect economic and environmental boundaries, and Chemical kinetics remains unchanged in microfl uidics, but medicinal product are particularly challenging in this respect. mass-transfer (diffusive domain) may be largely changed The adoption of optimal technology and innovation is in low volume reactors and may play a fundamental therefore a needed development. role on some reactions. Altogether, these parameters strongly infl uence reactions occurring at very low reagent concentrations. A successfully story is the application of microfl uidics and fl ow chemistry to the synthesis of radiotracers for Positron Emission Tomography (PET) (1).

The unique features of radiopharmaceutical chemistry PET radiotracers encompass an R&D fi eld with striking peculiarities (2). In this fi eld, three basic constraints limit the applicability of any chemical process: a) time, due to the short half-life of employed radionuclides; b) mass, due to the trace amounts of radioactive species and c) quality, since the fi nished product is for human use. Furthermore, safety plays a Figure 1. General chemical process scheme. stringent role due to the added risk from radiation exposure. Radiopharmaceuticals have a high added value in the biomedical scenario, as they allow to perform disease Microfl uidics and fl ow chemistry play an important role in this diagnosis and treatment on the basis of a molecular scenario, due to the fact that dominant physical quantities level understanding. This can be done even at an early (e.g. heat and mass transfer) tend to operate differently from disease stage, when more solutions may be available and conventional fl ask chemistry. Originally investigated in few practicable. To date, only a small number of compounds, industrial chemistry and academic environments, where it those fitting a market dimension that could support their benefi tted from the development of a dedicated microscale costly production, has reached an industrial scale.

28 Chimica Oggi - Chemistry Today - vol. 34(3) May/June 2016 2-[18F]fluoro-2-deoxyglucose ([18F]FDG) is (since decades) the work-horse of PET imaging tracers, with applications in cardiology, neurology and oncology. The widespread clinical availability of [18F]FDG resulted essential for its survival on the market, in spite of the fact that this tracer has high sensitivity but quite low specificity. Many other tracers have strived to reach the market and only a few have survived the development phase (3). The quest for radiopharmaceuticals with higher specificity for selected diseases would play a fundamental role in personalized and precision medicine (4), but a change in production paradigm would be required to obtain this endpoint in a sustainable manner. This is the point where the traditional vessel-based systems fall short of economical and environmental sustainability, and where new flow chemistry technologies can represent a game- Scheme 1. Radiofluorination reaction for 18[ F]T807 and [11C]MK-0233 changer. In this case, the use of microscale reactors may even be beneficial for the minimal amounts of reagent (radionuclides) and the time constraints (half-life) typical of Chemicals’ quantity reduction these processes. A common strategy with short half-life radiotracer is the The field has been recently reviewed (5) and many introduction of the labelling as the last step. This leads often examples are reported of the application of microfluidics to the use of precious and costly customized reagents. to radiopharmaceutical production. In here, we will focus In this case, the advantage of microfluidics relies on the on those particular benefits that, besides quicker and ability of handling small volumes of solutions, that can be more efficient processes, have also introduced chemical scaled down to few tens of nL (11). [18F]Fallypride, a D2 innovation and possibly paradigmatic changes. dopamine PET tracer, was efficiently prepared by using only 40mg of the tosylate precursor in microfluidics, with a 50-fold reduction versus the traditional 2mg that were necessary in OPPORTUNITIES vessel chemistry (12). Similarly, a custom microflow system was used to optimize the radiolabelling conditions (pH and Time reduction of radiolabelling reaction concentration) of an anti-Prostate Stem Cell Antigen diabody PET radionuclides can be regarded as precious but quickly with [18F]SFB (Scheme 2). Reaction scouting was performed expiring starting materials and time as a critical production in 120nL droplets holding as little as 50ng of the expensive parameter. Two of the most attractive (and used) precursor, thus achieving up to 2000-fold consumption radionuclides are 11C and 18F with half-lives of 20 and 110 reduction, compared to vessel methods. min, respectively (2). Therefore, reaction time and chemical reaction performances, dictate the production yields. The best way (easier chemical handling, better purity, higher yield and specific radioactivity) to produce18 F at a cyclotron is as fluoride ions, and nucleophilic fluorination is the target reaction with this radionuclide. Traditional vessel chemistry achieves satisfactory yields at 90-120°C, in polar aprotic solvents and in the presence of a base: typical reaction time ranges 5 to 30 minutes (6). However, a reaction Scheme 2. Radiolabelling of diabody with [18F]SFB. time <5 min would not be achievable in the 5-20 mL reactors, mainly because the modest heating efficiency is strongly dependent on the modality of energy delivery and Reduced amounts of precursors are linked to specific activity the vessel volume/shape (7). Furthermore, the unbalanced (the ratio of the radioactivity towards the moles of labelled and mass ratio between the radiolabelling agent and the unlabelled active molecule). High specific activity is associated precursor justify a pseudo-first order diffusion-controlled to a low mass, which means having both real tracer behaviour kinetics, which is penalized in vessel chemistry. and the absence of toxic and pharmacodynamic effects 18 Indeed, radiolabelling reactions in microfluidic reactors in vivo. Pike et al. have demonstrated (13) that [ F]XeF2, an generally occur with higher radionuclide incorporation in electrophilic radiofluorinating agent, can be produced with 18 - 19 reaction times <120 seconds, but typically in the 15-60 sec [ F]F by halogen exchange on [ F]XeF2 in CH3CN at high range. As an example, N. Vasdev et al. obtained similar temperatures. Thirty mg of precursor are required in a vessel results in the preparation of T807, a potential Alzheimer’s system, while 700 mg are sufficient in microfluidics, thus leading disease biomarker, when the reaction (Scheme 1) was a 30-fold higher specific activity. R.M. vanDam et al. (14) have conducted for 10 min at 130 °C in a 20 mL vessel (8) or by recently used an ElectroWetting On Dielectric (EWOD) chip using a 31.5 µL microreactor for just 45 sec at 210 °C (9). system, to reach the highest specific activity of 18[ F]Fallypride Similar results have also been obtained for the shorter (730 MBq/nmol) without starting from exceedingly high activity. half-life 11C, as in the case of [11C]MK-0233, in which the Interestingly, this result was possible due to the reduction of 19F Authors (10) reported the same incorporation yield for the isotopic dilution coming from the system adopted: 4µL droplets vessel reaction at 150°C for 6 min and for the microfluidic bearing 160 mg of the precursor which are exposed only to the reaction at 180°C for <10 sec. heating electrode area of <1 cm2.

Chimica Oggi - Chemistry Today - vol. 34(3) May/June 2016 29 Improved control streamline the development of new radiotracers by The basic parameters regulating the performance of speeding up the optimization of the radiolabelling condition. radiolabelling reactions in microfluidics are a) reactant By loading storage loops with reagents and delivering them quantities and mixing, b) heat transfer and temperature, in small boluses (few µLs), it is possible to give each reaction c) residence time (i.e. reaction time) and d) system its own “chemical history”. In a typical set-up, it is possible pressure. to run >30 different experiments by using few milligrams Aliquots of reactants, from few nL to µL, can be delivered of substrate per day and the same batch of radionuclide accurately, often as reaction plugs in a stream of inert precursors. With classic vessel-based radiochemistry the solvents, over a wide range of flow rates, fluidic design, same amount of precursors would allow 2-3 different in laminar or turbulent flow conditions. The high surface- reactions; therefore, accumulating >30 reactions would to-volume ratio featured by microfluidic systems, allows involve >15 days. Microfl uidic data on reaction conditions a fast and extremely accurate heating or cooling of can be used to plan optimization strategies (17) and scoping the reaction, with an outstanding homogeneity of further utilization (e.g. similar precursors). Scaling-up is also energy transfer throughout the microreactor. Finally, straightforward; for instance, the synthesis of the melanoma modifying the flow of reactants easily changes their PET tracer [18F]MEL050 (18), whose reaction conditions mixing ratio; while changing the overall flow is a simple were optimized at the 10µL scale, was directly scaled up and effective way to vary the time spent in the heated to a 100µL scale, maintaining its yield and specifi c activity zone. Finally, the microfluidic system can be pressurized, (comparable to the vessel-chemistry, but employing so that superheated solvents can be used and extended only 0.5mg of precursor vs. 5mg). This is possible since the reaction conditions easily attained. The net effect of the reaction conditions at the microfl uidic scale are exactly the improved reaction control is a high reaction accuracy same, independently from the total volume of the reagent and precision, which can lead to an improved process solutions passed through the system. selectivity but also to an easier technology transfer among different laboratories. Multi micro-batching (Dose On Demand, DOD) Reaction control is central in multiphase reactions. 11CO and The efficient use of radiopharmaceuticals strongly 11 11 CO2 are essential precursors in C-radiochemistry but depends upon minimizing the decay losses and efficient often poor results are obtained in gas-liquid processes. logistics and a timely utilization are mandatory; to date, K. Dahl et al. (15) generated a segmented flow by using the solution is to prepare large batches and dispense/ a solution of reagents and a catalyst (Scheme 3) and distribute them according to customers’ orders. Typically, small bubbles of [11C]CO to obtain a range of amides (> about 50% of the product is lost due to this organization. 10) by carbon monoxide insertion. In this work, they were The point-of-use approach enabled by microfluidic able to obtain a virtually quantitative trapping of the systems would allow to move the process closer to the radio-gas, yielding labelling efficiencies >40% with all the end-user and, at the same time, provide a chance of substrates. In particular, they obtained a 41% yield in the exploiting diverse microchips to separate the “chemical synthesis of [11C]raclopride, a useful D2 imaging agent. history” of a batch from the crude radionuclide precursor. The adaptation of the method to the different amines was Thus, in the shortest time possible, a radiotracer dose in the straightforward due to the outstanding reaction control, purity and formulation needed for the PET imaging scan mostly relying on the bubble size in the reagent solution would be available, almost in a DOD approach (Figure 2). stream. Our group reported the first proof of principle for DOD by producing 3 different [18F]fluoroalkyl cholines (19) using a two-steps process on the same microfluidic system.

Scheme 3. General reaction of [11C]CO insertive carbonylation.

Ease of automation Flow systems and micro-sized lab-on-chips, are natively controlled by automated interfaces, and electronic controls and sensors are commonly used. This generated some reluctance in being accepted in the standard chemical laboratory, in particular as an alternative to the round-bottom flasks. Conversely, radiochemistry is natively based on remote handling and automation has been adopted in radiochemical processing as early as Figure 2. Comparison of centralized distribution and dose on demand (DOD). On the left side, in the traditional centralized possible (16). This favourable ground has sped up the batch approach, one central radiopharmacy will produce the application of microfluidic techniques to the production starting raw activity and also transform it (red arrow) in a big batch of fi nished radiopharmaceutical, thus distributing to the various of radiopharmaceuticals. users (i.e. hospitals, H) the same molecule. On the right side, in the DOD approach, one central site produces the starting raw activity Fast radiolabelling optimization and distribute it to various users, where miniaturized DOD systems (green arrow) will allow to produce the different tracers needed by Perhaps the most useful use of microfl uidics in the fi nal users. radiopharmaceutical chemistry, is the possibility to

30 Chimica Oggi - Chemistry Today - vol. 34(3) May/June 2016 Single-use systems solution) can be concentrated with PET radionuclides, Point-of-care diagnostics have deeply changed patients’ whose extremely low chemical concentration make and disease management, but the need of harsher the incorporation yield relatively unrelated to the reaction conditions and more aggressive chemical amount of starting activity. Another solution, still not reagents complicate the realization of a similar approach employed in radiochemistry, would be the use of process for radiopharmaceuticals. The EWOD based systems are parallelization, potentially implementing different currently the closest paradigm approaching a full lab-on- productions on each parallel channel. chip (20). This prototype is able to perform multi-step/one- pot reactions in a controllable manner by moving discrete Microreactor interfacing droplets of reagents without any tubing or valve, but using As radiopharmaceutical lab-on-chip are still complex a set of electrodes. However, the interface with a micro- and usually process-dedicated, it is often necessary to sized purification system and scaling up solutions are still to interface the reactor to the classic labware (tubings, be fully developed. pumps, reservoirs) and couple to heating systems and Radiopharmaceuticals based on peptide moieties and sensors. For instance, the connection options vary from radiometal labelling (e.g. 64Cu, 68Ga, 89Zr, [18F]AlF, 99mTc), orthogonal (Futurechemistry), in-line connections (Advion comprise radionuclides not requiring a cyclotron and the NanoTek), plug-and-play (Micronit), finger-tight (Little use of milder reaction conditions and simpler purifications. Things Factory) and custom (Syrris), without demonstration PDMS, a very flexible and cheap material, has been of a sensible advantage over the others. This problem used to build chips that proved successful in several PET and the variability of other components that constitute radiometals’ labelling reactions (21). the whole radiochemistry system have suggested the These two examples could conceivably be disposable for construction of modular systems (23) as the optimal producing human doses, and may bring extensive gain choice to ensure an adequate flexibility of use. on the side of regulations compliance and economic/ operational planning. Materials A big issue (opportunity and threat) is the effect of surface interactions, due to the high surface/volume ratio CHALLENGES featured by microchannel reactors. This is particularly important for PET radiopharmaceutical chemistry, in which Traditional vessel approach philosophy radionuclide are at ultra-trace levels, and losses due to The round bottom flask is still the first choice in Academia. adsorption may easily occur, particularly when using [18F] Conversely, the industry has paid more attention to F- and glassy materials. Very few papers have studied (micro)flow chemistry; in particular for exothermic and the compatibility of materials with PET radionuclides in radical reactions or greener processes. The micro-sized their original form (24), while the losses of radioactivity in chemistry seems to have levelled off after the initial enthusiasm. However, the growth of industries dedicated to the manufacturing of micro-sized devices (e.g. Syrris, Little Things Factory, Micronit, Futurechemistry) can both facilitate and promote their acceptance by academic and R&D groups (22). Although some brilliant examples, like EWOD, triggered the attention on microdevices, much has to be done to fully transfer this approach to the everyday chemistry and to overcome some issues, still causing puzzling and attrition among users. This fact is present in the radiochemistry field as well, were the need to renew the currently widely utilized vessel system is limited, and microfluidic system (where available) are regarded as a choice for research and not routine activities. Perhaps, an increased number of publications on radiotracers produced by microfluidic devices and used for human studies would slowly shift the attention towards this innovative and promising approach.

Reaction (residence) vs. processing time In microfluidic flow systems, there is a marked difference between residence time, which replaces the reaction time in vessel-chemistry, and processing time, which is dictated by the time necessary to have all the reactant mixture passed through the reactor; the latter may be much longer than the former. Possible solutions would be to use longer reactors or concentrate the reactants (if possible) and will depend on process characteristics (backpressure, laminar/turbulent flow, mass-transfer, etc). Fortunately, the major reactant (i.e. the radionuclide labelling commercial microfluidic systems is sometimes marginally The down-scale impulse, which has started from reported (25). Materials’ features may also change electronics, is moving on to chemistry and the application with aging (e.g. the creation of nano-sized surface of these micro-sized techniques would be incredibly irregularities) so that radioactivity recovery may change helpful, especially in the particularly challenging field with the time. The recent availability of a 3D printer, of radiochemistry. The exploration of chemistry at the which can use resins to build small chips (Fluidic Factory, microscale is a new challenge: this effort, along with the Dolomite, UK), will hopefully open up the way to new multidisciplinary contribution by the nanomaterial science, materials. 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