Simplified and Highly-reliable automated production of [18F]FSPG for clinical studies

Background (S)-4-(3-18F-Fluoropropyl)-L-Glutamic Acid ([18F]FSPG) is a positron emission tomography (PET) tracer that specifically targets the cystine/glutamate antiporter (xc-), which is frequently overexpressed in cancer and several neurological disorders. Pilot studies examining the dosimetry and biodistribution of ([18F]FSPG in healthy volunteers and tumor detection in patients with non-small cell lung cancer, hepatocellular carcinoma, and brain tumors showed promising results. In particular, low background uptake in the brain, lung, liver, and bowel was observed that further leads to excellent imaging contrasts of [18F]FSPG PET. However, reliable production-scale cGMP-compliant automated procedures for [18F]FSPG production are still lacking to further increase the utility and clinical adoption of this radiotracer. Herein, we report the optimized automated approaches to produce [18F]FSPG through two commercially available radiosynthesizers capable of supporting centralized and large-scale production for clinical use. Results Starting with activity levels of 60–85 GBq, the fully-automated process to produce [18F]FSPG took less than 45 minutes with average radiochemical yields of 22.56 ± 0.97% and 30.82 ± 1.60% (non-decay corrected) using TRACERlab™ FXFN and FASTlab™, respectively. The radiochemical purities were > 95% and the formulated [18F]FSPG solution was determined to be sterile and colorless with the pH of 6.5–7.5. No radiolysis of the product was observed up to 8 hours after final batch formulation. Conclusions In summary, cGMP-compliant radiosyntheses and quality control of [18F]FSPG have been established on two commercially available synthesizers leveraging high activity concentration and radiochemical purity. While the clinical trials using [18F]FSPG PET are currently underway, the automated approaches reported herein will accelerate the clinical adoption of this radiotracer and warrant centralized and large-scale production of [18F]FSPG.

effectiveness of [ 18 F]FDG PET, and increasingly, PET tracers targeting very speci c molecular and even 'druggable' pathways are required. As a result, there is a great demand for additional radiopharmaceuticals with unique targeting capability for molecular imaging.
Amino acids play a variety of critical roles in many cellular functions. Due to an accelerated growth rate in cancer cells, the demand for amino acids is elevated as well. Consequently, imaging radiopharmaceuticals based on radiolabeled amino acid analogues have long been appreciated to provide information regarding protein metabolism of malignant cells. Recently, a glutamate-based tracer, (S)-4-(3-[ 18 F] uoropropyl)-L-glutamic acid ([ 18 F]FSPG), has started to receive great attention as it has been shown to speci cally target the cystine/glutamate antiporter (xc − ). The xc − antiporter is a plasma membrane transporter mediating cellular uptake of cystine in exchange for intracellular glutamate with an equal stoichiometry ratio (Lo, Ling, Wang, & Gout, 2008). Although cysteine plays important roles in protein synthesis and in maintaining redox balance, de novo biosynthesis or a catabolic supply of cysteine is not su cient to meet the high demand for antioxidant synthesis under conditions of oxidative stress such as in cancer and neurological disorders, which drives xc-activity to ensure cysteine supply Producing radiotracers with highly consistent radiochemical yield and quality is required to bring promising radiotracers from bench to bedside. Although several groups have described automated procedures to produce [ 18 F]FSPG, previous reports were focused on small-scale production (less than 400 MBq of the nal product) and with complex modi cations of commercially available cartridges (  mL SPE tubes were obtained from Millipore Sigma (Burlington, MA). Acrodisc® glass membrane lter and sterilized water for injection (SWI) were products of PALL Corp. and Hospira, respectively. All other references of 'water' refers to Milli-Q water (18 MΩ•cm) taken from a Millipore Milli-Q Integral 5 water puri cation system and were primarily used in quality control processes. Anhydrous acetonitrile (99.8%) used in the evaporation and labeling steps was from Sigma-Aldrich. Other reagents used in the production process of [ 18 F]FSPG include sodium hydroxide (4M NaOH, RICCA), sulfuric acid (1M H 2 SO 4 , Fisher), ethanol (Pharmco), sodium phosphate dibasic dihydrate (Acros Organics); in addition, potassium carbonate and sodium chloride were purchased from Thermo Fisher.
The Krypto x stock solution was prepared, in-house, by adding 94.0 ± 1.0 mg of Krypto x (Cryptand-222) and 9.40 ± 0.10 mg of potassium carbonate in a glass container. The solid mixture was then dissolved with 5 mL of SWI and 5 mL of acetonitrile. The phosphate buffered solution used for nal elution of [ 18 F]FSPG contained 603 ± 10 mg of sodium chloride and 700 ± 10 mg of sodium phosphate dibasic dihydrate that were dissolved by 100 mL of SWI. All chemicals were used without further puri cation. Nitrogen and argon gas used primarily in drying and transferring of solutions were provided through Matheson Tri-gas. The automation synthesis on the TRACERlab™ FXFN and FASTlab™ modules were controlled by the TRACERLab FX software and the FASTlab Developer software, respectively. F]Fluoride from the cartridge into the reaction vessel. The solution was heated to 120°C to remove the water and acetonitrile initially. Next, the precursor for the reaction, (2S,4S)di-tert-butyl 2-(3((naphthalene-2-ylsulfonyl)oxy)propyl)-4-(tritylamino)pentanedioate dissolved in anhydrous acetonitrile, was added to the reaction vessel. The reaction was heated at 105°C for 5 minutes in a closed reaction vessel to promote the substitution of the 18 F for the sulfonate leaving group. Next, the 1M sulfuric acid solution was added and the reaction was heated at 105°C for 4 minutes to reveal the carboxylic acid groups. Hydrolysis occured as 4.0M sodium hydroxide was added and the reaction was heated to 70°C for a period of 5 minutes. The solution was then allowed to cool and a second batch of the 1M sulfuric acid was added to acidify the mixture. The reaction mixture was transferred onto the MCX cartridges connected in series.   Table 1. At position 9, a pre-conditioned QMA cartridge was installed and used as purchased. Two Oasis® MCX cartridges were attached and conditioned with 10 mL of the formulated phosphate buffered saline, followed by 10 mL of air, then assembled to a single glass membrane lter, as demonstrated in Fig. 5a, and connected to the lines at position 10. To prepare the Alumina N Cartridge/Superclean™ ENVI-Carb™ assembly for the radiosynthesis, approximately 1.5 grams of ENVI-Carb resin was weighed and added to a 6 mL Supelco tube and a frit was packed in to secure the resin. The column was then sequentially conditioned with 10 mL of ethanol, followed by 10 mL of the formulated phosphate buffered saline, then dried with 10 mL of air. An Alumina N Plus Long SepPak cartridge was activated with 10 mL of SWI followed by 10 mL of air. The Alumina cartridge was then attached to the top of the ENVI-Carb column, as illustrated by Fig. 5b, and then assembled to the position 11 on the TRACERlab™ FXFN module. A nal product vial was connected to the outgoing transfer line of the double-neck vial, as shown in position 12.   grams of ENVI-Carb resin was weighed and added to a 6 mL Supelco tube and a frit was packed in to secure the resin. The column was then sequentially conditioned with 10 mL of ethanol, followed by 10 mL of the formulated phosphate buffered saline, then dried with 10 mL of air. The inlet of the column was connected to the tubing attached to the cassette position CP23. The outlet of the column was connected with the transfer line that would eventually delivered the puri ed [ 18 F]FSPG for injection to the nal product vial.  Table 3. The average non-decay-corrected radiochemical yield was 22.56 ± 0.97% with radiochemical purity of 96.31 ± 0.62% when less than 37 GBq of the starting activity and 6 mg of the precursor were applied. However, dramatical reduction in radiochemical yield and slightly lower radiochemical purity were observed when larger starting activity was used. This limit could further be resolved by adding additional amounts of the precursor and ENVI-Carb graphitized carbon for the reaction and nal puri cation process. Because our ultimate goal was to facilitate the centralized production of [ 18 F]FSPG for clinical use, we translated these ndings to FASTlab™, a cassette-based module, for easier adoption by other radiopharmaceutical manufacturing facilities.  Table 4. While a consistently high radiochemical purity (95.96 ± 0.70%) was maintained, the average radiochemical yields was also observed to be signi cantly higher than from the TRACERlab counterpart, 30.82 ± 1.60% vs. 22.56 ± 0.97%, p < 0.0001. The much improved radiochemical yields from the FASTlab, in addition to changes previously mentioned, can also be attributed to the compact design of the reactor and more e cient built-in vacuum system in the module compared to the counterpart TRACERlab. Radio-HPLC analysis of the produced [ 18 F]FSPG for injection showed > 95% radiochemical purity. Further analysis after 8h con rmed that the product remained stable (Fig. 10). The molar activity calculated from the activity yields at EOS and FSPG was in the range of 100-292.3 GBq/µmol. The residual ethanol and acetonitrile concentrations were less than 0.2% and 0.005% (v/v), respectively. The pH was 6.5 to 7.5 and the bubble point was 70 to 75 psi. In addition, the sterility, pyrogenicity, and all other quality control tests passed speci cations based on the USP < 823 > guidelines.

Discussion
In

Declarations
Availability of data and materials All data generated or analyzed during this study are included in this manuscript.

Contributions
MLC and RTTA designed, performed and analyzed the experiments. HCM evaluated the results concerning clinical applications. All authors read and approved the nal manuscript.
HCM is a CPRIT Scholar of Cancer Research.       Five (5) point calibration of FSPG reference standard complexation with OPA reagent by HPLC analysis. The blue regression curve represents the molar concentration, whereas the red curve represents the mass concentration, measured against the peak area.