On-demand radiosynthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) on an electrowetting-on-dielectric microfluidic chip for 18F-labeling of protein

An all-electronic, droplet-based batch microfluidic device, operated using the electrowetting on dielectric (EWOD) mechanism was developed for on-demand synthesis of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), the most commonly used 18F-prosthetic group for biomolecule labeling. In order to facilitate the development of peptides, and proteins as new diagnostic and therapeutic agents, we have diversified the compact EWOD microfluidic platform to perform the three-step radiosynthesis of [18F]SFB starting from the no carrier added [18F]fluoride ion. In this report, we established an optimal microliter droplet reaction condition to obtain reliable yields and synthesized [18F]SFB with sufficient radioactivity for subsequent conjugation to the anti-PSCA cys-diabody (A2cDb) and for small animal imaging. The three-step, one-pot radiosynthesis of [18F]SFB radiochemistry was adapted to a batch microfluidic platform with a reaction droplet sandwiched between two parallel plates of an EWOD chip, and optimized. Specifically, the ratio of precursor to base, droplet volume, reagent concentration, reaction time, and evaporation time were found be to be critical parameters. [18F]SFB was successfully synthesized on the EWOD chip in 39 ± 7% (n = 4) radiochemical yield in a total synthesis time of ∼120 min ([18F]fluoride activation, [18F]fluorination, hydrolysis, and coupling reaction, HPLC purification, drying and reformulation). The reformulation and stabilization step for [18F]SFB was important to obtain a high protein labeling efficiency of 33.1 ± 12.5% (n = 3). A small-animal immunoPET pilot study demonstrated that the [18F]SFB-PSCA diabody conjugate showed specific uptake in the PSCA-positive human prostate cancer xenograft. The successful development of a compact footprint of the EWOD radiosynthesizer has the potential to empower biologists to produce PET probes of interest themselves in a standard laboratory.

[1]  R. M. van Dam,et al.  Ultra-compact, automated microdroplet radiosynthesizer. , 2019, Lab on a chip.

[2]  R. Tavaré,et al.  18F-labeled anti-human CD20 cys-diabody for same-day immunoPET in a model of aggressive B cell lymphoma in human CD20 transgenic mice , 2018, European Journal of Nuclear Medicine and Molecular Imaging.

[3]  A. Wu,et al.  Dual-Modality Immuno-PET and Near-Infrared Fluorescence Imaging of Pancreatic Cancer Using an Anti–Prostate Stem Cell Antigen Cys-Diabody , 2018, The Journal of Nuclear Medicine.

[4]  M. Phelps,et al.  Performing radiosynthesis in microvolumes to maximize molar activity of tracers for positron emission tomography , 2018, Communications Chemistry.

[5]  R. Tavaré,et al.  ImmunoPET of Malignant and Normal B Cells with 89Zr- and 124I-Labeled Obinutuzumab Antibody Fragments Reveals Differential CD20 Internalization In Vivo , 2017, Clinical Cancer Research.

[6]  H. Saji,et al.  Continuous-Flow Synthesis of N-Succinimidyl 4-[18F]fluorobenzoate Using a Single Microfluidic Chip , 2016, PloS one.

[7]  Sean M. Preshlock,et al.  (18)F-Labeling of Arenes and Heteroarenes for Applications in Positron Emission Tomography. , 2016, Chemical reviews.

[8]  G. Sonn,et al.  Fluorescent Image–Guided Surgery with an Anti-Prostate Stem Cell Antigen (PSCA) Diabody Enables Targeted Resection of Mouse Prostate Cancer Xenografts in Real Time , 2015, Clinical Cancer Research.

[9]  Amanda C. Freise,et al.  In vivo imaging with antibodies and engineered fragments. , 2015, Molecular immunology.

[10]  Xiaoyuan Chen,et al.  Fluorine-18 Radiochemistry, Labeling Strategies and Synthetic Routes , 2014, Bioconjugate chemistry.

[11]  J. Collins,et al.  Fully Automated Production of Diverse 18F-Labeled PET Tracers on the ELIXYS Multireactor Radiosynthesizer Without Hardware Modification , 2014, The Journal of Nuclear Medicine Technology.

[12]  Mark Lazari,et al.  Radiolabelling diverse positron emission tomography (PET) tracers using a single digital microfluidic reactor chip. , 2014, Lab on a chip.

[13]  Johannes Czernin,et al.  Efficient Radiosynthesis of 3′-Deoxy-3′-18F-Fluorothymidine Using Electrowetting-on-Dielectric Digital Microfluidic Chip , 2014, The Journal of Nuclear Medicine.

[14]  Markus J. Bröcker,et al.  Titelbild: Umkodierung des genetischen Codes mit Selenocystein (Angew. Chem. 1/2014) , 2014 .

[15]  Jeffrey Collins,et al.  High yield and high specific activity synthesis of [18F]fallypride in a batch microfluidic reactor for micro-PET imaging. , 2014, Chemical communications.

[16]  Paul Watts,et al.  Radiochemistry on chip: towards dose-on-demand synthesis of PET radiopharmaceuticals. , 2013, Lab on a chip.

[17]  P. Watts,et al.  Positron Emission Tomography Radiosynthesis in Microreactors , 2012, Journal of Flow Chemistry.

[18]  Saman Sadeghi,et al.  Micro-chemical synthesis of molecular probes on an electronic microfluidic device , 2011, Proceedings of the National Academy of Sciences.

[19]  A. Scott,et al.  A simplified protocol for the automated production of succinimidyl 4‐[18F]fluorobenzoate on an IBA Synthera module , 2011 .

[20]  H. Kolb,et al.  Batch‐mode microfluidic radiosynthesis of N‐succinimidyl‐4‐[18F]fluorobenzoate for protein labelling , 2011 .

[21]  Ganghua Tang,et al.  A facile automated synthesis of N‐succinimidyl 4‐[18F]fluorobenzoate ([18F]SFB) for 18F‐labeled cell‐penetrating peptide as PET tracer , 2010 .

[22]  A. Wu,et al.  Positive progress in immunoPET--not just a coincidence. , 2010, Cancer biotherapy & radiopharmaceuticals.

[23]  Anna M Wu,et al.  Antibody vectors for imaging. , 2010, Seminars in nuclear medicine.

[24]  F. Salazar,et al.  An affinity matured minibody for PET imaging of prostate stem cell antigen (PSCA)-expressing tumors , 2010, European Journal of Nuclear Medicine and Molecular Imaging.

[25]  Gaurav J Shah,et al.  Supplementary Material (esi) for Lab on a Chip Eletronic Supplementary Information for B823541d: Fluidic Conduits for Highly Efficient Purification of Target Species in Ewod-driven Droplet Microfluidics 1. Sample Purification by Serial Dilution , 2022 .

[26]  Arkadij M Elizarov,et al.  Microreactors for radiopharmaceutical synthesis. , 2009, Lab on a chip.

[27]  Wenbin Zeng,et al.  Facile synthesis of N‐succinimidyl 4‐[18F]fluorobenzoate ([18F]SFB) for protein labeling , 2008 .

[28]  Haoran Sun,et al.  Competitive demethylation and substitution in N,N,N-trimethylanilinium fluorides , 2007 .

[29]  J. Sutcliffe,et al.  Fully automated preparation of n.c.a. 4-[18F]fluorobenzoic acid and N-succinimidyl 4-[18F]fluorobenzoate using a Siemens/CTI chemistry process control unit (CPCU). , 2007, Applied radiation and isotopes : including data, instrumentation and methods for use in agriculture, industry and medicine.

[30]  M. Zalutsky,et al.  Synthesis of N-succinimidyl 4-[18F]fluorobenzoate, an agent for labeling proteins and peptides with 18F , 2006, Nature Protocols.

[31]  Monica Brivio,et al.  Miniaturized continuous flow reaction vessels: influence on chemical reactions. , 2006, Lab on a chip.

[32]  F. Füchtner,et al.  Module-assisted synthesis of the bifunctional labelling agent N-succinimidyl 4-[(18)F]fluorobenzoate ([(18)F]SFB). , 2005, Applied radiation and isotopes : including data, instrumentation and methods for use in agriculture, industry and medicine.

[33]  R. Bergmann,et al.  Radiolabelling of isopeptide Nε-(γ-glutamyl)-l-lysine by conjugation with N-succinimidyl-4-[18F]fluorobenzoate , 2003 .

[34]  Sanjiv Sam Gambhir,et al.  AMIDE: a free software tool for multimodality medical image analysis. , 2003, Molecular imaging.

[35]  S. Cho,et al.  Creating, transporting, cutting, and merging liquid droplets by electrowetting-based actuation for digital microfluidic circuits , 2003 .

[36]  Paul Watts,et al.  Micro reactors: principles and applications in organic synthesis , 2002 .

[37]  Okarvi Sm,et al.  Recent progress in fluorine-18 labelled peptide radiopharmaceuticals , 2001 .

[38]  S. Okarvi,et al.  Recent progress in fluorine-18 labelled peptide radiopharmaceuticals , 2001, European Journal of Nuclear Medicine.

[39]  D. Hwang,et al.  A new method for the NCA production of [18F]fluoromethane , 1994 .

[40]  W. Bannwarth,et al.  Formation of carboxamides with N,N,N′,N′-tetramethyl (succinimido) uronium tetrafluoroborate in aqueous / organic solvent systems , 1991 .