Optimization of Radiochemical Reactions using Droplet Arrays
February 12th, 2021
This method describes the use of a novel high-throughput methodology, based on droplet chemical reactions, for the rapid and economical optimization of radiopharmaceuticals using nanomole amounts of reagents.
Current automated radio synthesizers are designed to produce large batches of widely used radiopharmaceuticals, such as FDG. Due to the limited number of syntheses possible per day, and relatively high reagent consumption, these systems, however, are not well-suited for performing synthesis optimization studies. With this technique, throughput is significantly increased by performing up to 16 simultaneous reactions in parallel and reagent consumption is reduced a hundred-fold.
Furthermore, by performing reactions in parallel, fair batches of radioisotope are needed to complete a study. Increased throughput enables wider exploration of reaction conditions with greater number of replicates each compared to the use of conventional instruments. While this protocol shows the optimization of precursor concentration in the synthesis of fallypride, the technique can be used to optimize other conditions and other radiopharmaceuticals.
Begin by fabricating batches of multi-reaction microdroplet chips from four inch silicon wafers using standard photolithography techniques. In this protocol, the high-throughput optimization of precursor concentration is demonstrated with the synthesis of the radiopharmaceutical Fallypride. 16 simultaneous reactions can be performed on a single chip.
The conditions to be compared are mapped to the reaction sites. Prepare a stock solution of the reaction solvent consisting of thexyl alcohol and acetonitrile in a one-to-one by volume mixture. Ensure that the volume is enough to create the planned dilution series.
Prepare a 30 microliters stock solution of precursor in the reaction solvent with the maximum concentration to be explored. From the precursor stock solution and reaction solvent perform two times serial dilutions in a set of microcentrifuge tubes to prepare the different concentrations of the precursor solution. Prepare another set of microcentrifuge tubes to collect each crude reaction product using a permanent marker to label each tube with a unique number.
Ensure that the total number of microcentrifuge tubes matches the number of conditions multiplied by the number of replicates. Prepare a 10 milliliter stock of collection solution comprising of nine to one methanol to deionized water. Aliquot 50 microliters of each into an additional set of 16 microcentrifuge tubes labeled as collection solution.
Prepare a Fluoride stock solution by mixing around seven millicuries of the Fluoride source with 56 microliters of 75 millimolar tetrabutylammonium bicarbonate and diluting with DI water up to 140 microliters. Using a micropipette, load an eight microliter droplet of Fluoride stock solution on the first reaction spot of a multi-reaction chip. Measure the activity of the chip by placing it in a dose calibrator and record the time at which measurement is conducted.
Remove the chip from the dose calibrator and load an eight microliter droplet of Fluoride stock solution on the second reaction spot. Measure the activity on the chip and record the time at which measurement is conducted. Repeat this process for all other reaction sites.
Calculate the activity loaded per reaction spot by taking the activity measurement after loading the radioisotope and subtracting the previous measurement before that site was loaded. To align the multi-reaction chip on the heater, add a thin layer of thermal paste on top of the ceramic heater. Carefully place the chip on top of the heater using tweezers aligning the reference corner of the chip with the reference corner of the heater.
The chip should overhang the heater by a small amount. Heat the chip for one minute by setting the heater to 105 degrees Celsius in the control program to evaporate the droplets, leaving a dried residue of Fluoride and tetrabutylammonium bicarbonate. Then, cooled the chip by setting the heater to 30 degrees Celsius and turning on the cooling fan with the control program.
Using a micropipette, add a six microliters solution of fallypride precursor on top of the dried residue on the first reaction site. Repeat this for all other reaction sites on the chip. Use the optimization plan to determine which concentration of the dilution series is used for each reaction site.
Heat the chip to 110 degrees Celsius for seven minutes using the control program to perform the radiofluorination reaction, then cool the chip by setting the heater to 30 degrees Celsius and turning on the cooling fan with the control program. Collect the crude product at the first reaction site by adding 10 microliters of collection solution from the designated microcentrifuge tube. After waiting for five seconds, aspirate the diluted crude product and transfer it to the corresponding collection microcentrifuge tube.
Repeat this process a total of four times using the same pipette tip and then close the microcentrifuge tube. Repeat these steps to collect the crude product from all other reaction sites on the chip. To determine the collection efficiency for the first reaction on the chip, place the microcentrifuge tube with the collected crude product of the first reaction spot in the dose calibrator to measure the activity.
Record the measurement and time of the measurement. Repeat this process for each of the collected crude products. Calculate the collection efficiency by dividing the activity of the collected crude product by the starting activity measured for the same reaction site.
Repeat this for all other reaction sites on the chip. Next, analyze the composition of each collected crude product. With a pencil, draw a line 15 millimeters away from the bottom edge of the TLC plate and another line 50 millimeters away from the same edge.
The first line is the origin line and the second is the solvent frontline. Draw eight small X's along the origin line at five millimeter spacing to define the sample spotting position for each of the eight lanes. Using a micropipette, transfer 0.5 microliters of the first crude product onto the TLC plate at the X for the first lane.
Repeat this for additional crude products, then wait for the spots to dry. Develop each TLC plate using a mobile phase of 60%of acetonitrile in 25 millimolar ammonium formate with 1%TEA until the solvent front reaches the solvent frontline. At that time, remove the TLC plate from the chamber and wait for the solvent on the TLC plate to dry, then placed the TLC plate in the Cerenkov imaging system and cover it with a glass microscope slide.
Obtain a radioactivity image of each TLC plate by setting the Cerenkov imaging system to a five minute exposure, then select the produced file of the image to TLC plate and perform standard image corrections. Use region of interest analysis for the first lane of the first TLC plate. Draw regions around each band visible in the lane, then compute the fraction of integrated intensity of each region compared to the total integrated intensity of all regions.
Determine the fluorination efficiency as the fraction of activity in the Fallypride band. Repeat this analysis for all other lanes on all TLC plates. Then, compute the crude radiochemical yield for each reaction and choose the optimal precursor concentration by examining the crude radiochemical yield as a function of precursor concentration.
Optimization studies of the radiopharmaceutical Fallypride were performed by varying precursor concentration in thexyl alcohol acetonitrile. Reactions were performed at 110 degrees Celsius per seven minutes. Collection efficiency and sample composition are shown here.
The fluorination efficiency increased with increasing precursor concentration and the concentration of unreacted Fluoride decreased. There was a small amount of radioactive side product at low precursor concentrations, which was not formed at the higher precursor concentrations. The collection efficiency was nearly quantitative for most conditions, though it dropped slightly at low precursor concentrations.
The highest crude radiochemical yield was achieved with a 39 millimolar precursor concentration. At this condition, the fluorination efficiency was 96%The crude RCY was 87%and there was no observed radioactive side product formation. It is critical to have a map plan of what reaction condition corresponds to which reaction droplet on the chip and to have appropriately labeled reagent tubes and product collection tubes that can be double-checked during the experiment.
The procedure can be used for the optimization of other reaction conditions, such as amount of base, type of solvent, or reaction volume. It can also be used to optimize the synthesis of other radiopharmaceuticals.
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Overview
This article presents a high-throughput platform for optimizing radiopharmaceutical synthesis using parallel miniature droplet-based reactions on patterned polytetrafluoroethylene-coated silicon chips. The method enables rapid screening of reaction parameters with minimal reagent consumption, significantly increasing throughput compared to conventional automated radiosynthesizers. The protocol is demonstrated through the optimization of precursor concentration in the synthesis of the radiopharmaceutical Fallypride.
Key Study Components
Area of Science
- Radiochemistry
- Radiopharmaceutical development
- High-throughput synthesis
Background
- Conventional automated radiosynthesizers are designed for large-scale clinical production.
- These systems are inefficient for reaction optimization due to high reagent consumption and limited daily throughput.
- Contamination and radioactive decay requirements further slow down iterative studies.
- There is a need for platforms that allow rapid, parallel optimization of radiochemical reactions with reduced reagent use.
Purpose of Study
- To develop and demonstrate a platform for performing arrays of miniature, parallel radiochemical reactions.
- To enable rapid optimization of reaction parameters such as precursor concentration, solvent, temperature, and time.
- To reduce reagent consumption and increase experimental throughput in radiopharmaceutical development.
Methods Used
- Fabrication of multi-reaction microdroplet chips from silicon wafers using photolithography.
- Parallel loading of reaction sites with fluoride stock solution and precursor solutions at varying concentrations.
- Controlled heating and cooling of chips to perform radiofluorination reactions.
- Collection and analysis of crude products using thin-layer chromatography (TLC) and Cerenkov imaging.
- Calculation of collection efficiency and radiochemical yield for each reaction condition.
Main Results
- Up to 16 simultaneous reactions can be performed on a single chip, reducing reagent consumption by a hundred-fold.
- Fluorination efficiency increased with higher precursor concentrations, while unreacted fluoride and side products decreased.
- The highest crude radiochemical yield (87%) and fluorination efficiency (96%) were achieved at 39 mM precursor concentration, with no observed radioactive side products.
- Collection efficiency was nearly quantitative for most conditions, with slight decreases at low precursor concentrations.
Conclusions
- The chip-based platform enables rapid, high-throughput optimization of radiochemical reactions with minimal reagent use.
- This approach allows for efficient exploration of reaction parameters and can be adapted for other radiopharmaceuticals and reaction conditions.
- Proper mapping and labeling of reaction conditions and collection tubes are critical for experimental accuracy.
Frequently Asked Questions
Executive Industry Relevance
High-throughput optimization of radiochemical reactions is a critical bottleneck in radiopharmaceutical discovery, where reagent cost, radioactive contamination, and instrument downtime limit experimental cycles. The droplet array chip platform enables parallel, miniaturized reaction screening, accelerating parameter optimization and reducing resource consumption. This capability enhances predictive confidence and supports rapid iteration at the early discovery and lead identification stages for novel radiotracer development.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables systematic interrogation of reaction parameters for new radiotracer candidates.
- Supports functional validation of synthetic routes with minimal radioactive waste.
- Facilitates rapid de-risking of chemical feasibility in target validation workflows.
- Improves predictive confidence for advancing promising radiopharmaceuticals.
Screening & Assay Development
- Prepares validated radiochemical conditions for downstream biological assays.
- Standardizes reaction outputs for reproducible compound evaluation.
- Enables scalable, parallel screening of multiple reaction variables.
- Reduces reagent and isotope consumption, supporting sustainable assay development.
Translational & Preclinical Research
- Aligns optimized radiochemical synthesis with preclinical imaging and biodistribution studies.
- Ensures continuity from discovery chemistry to translational radiotracer evaluation.
- Supports risk-adjusted advancement of candidates with robust synthetic profiles.
- Provides mechanistic insight into reaction efficiency and side product formation.
Pipeline & Workflow Integration
This droplet array platform fits at the interface of early discovery and lead optimization, bridging chemical feasibility with translational readiness for radiopharmaceuticals.
- Discovery Biology: Accelerates hypothesis testing by enabling rapid, parallel optimization of synthetic parameters.
- Screening: Delivers reproducible, quantitative outputs for comparing reaction conditions and yields.
- Analytics: Provides standardized measurements of radiochemical yield and efficiency for data-driven decision making.
- Translational Research: Facilitates seamless transition from optimized synthesis to preclinical validation studies.
- Enterprise Reuse: Establishes a reusable platform for iterative optimization across diverse radiotracer programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in radiochemical synthesis.
- Operational Value: Standardizes and scales reaction optimization with minimal reagent and isotope use.
- Strategic Value: Enables faster go/no-go decisions and reduces late-stage risk in radiopharmaceutical portfolios.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of high-potential radiotracer candidates.
Implementation Considerations
- Requires expertise in radiochemistry and microfluidic chip handling.
- Needs access to dose calibrators, TLC, and Cerenkov imaging systems for analysis.
- Demands rigorous mapping and labeling for cross-team reproducibility.
- Adaptable to various reaction conditions and radiotracer chemistries.
- Limited by the number of parallel reactions per chip and analytical throughput.
Biopharma FAQs
Chapters in this video
0:05
Introduction
1:00
Planning and Optimizing the Study
1:37
Preparation of Reagents and Materials for Optimizing the Radiosynthesis of [18F]Fallypride
3:11
Parallel Synthesis of [18F]Fallypride with Different Precursor Concentrations
6:02
Synthesis Analysis to Determine Reaction Performance and Optimal Conditions
8:55
Results: Influence of Precursor Concentration on the Microdroplet Synthesis of [18F]Fallypride
10:08
Conclusion
