Automated Radiosynthesis of [¹⁸F]DPA-714 on a Commercially Available IBA Synthera^®

Abstract

The goal of this work was to develop a reliable method to produce the well-validated microglial activation PET tracer, [¹⁸F]DPA-714, routinely for clinical and preclinical research using an IBA Synthera^®. Optimization of literature methods included reduced precursor mass and use of TBA HCO₃ as the phase transfer agent in place of Kryptofix^® 222 in a 65-minute synthesis with an average activity yield of 24.6 ± 3.8% (n = 5). Successful quality control testing and process validation results are reported.

1. Introduction

Neuroinflammation is an adaptive response of central nervous system (CNS) tissue to various injurious stimuli and can be triggered by either endogenous or exogenous factors. In response to neuronal insult, a multitude of cellular and molecular processes, primarily associated with microglia and astrocyte activation, macrophage transport, cytokine and chemokine release, and maintenance of the blood brain barrier, ensue to restore homeostasis.(Shields et al., 2020) In addition, there are consequences of chronic insults such as damage to neurons and demyelination.(Kwon and Koh, 2020) Radiation therapy, as experienced by many pediatric patients during treatment for brain tumors, can result in neuroinflammation and potential undesired side-effects in these patients.(Lumniczky et al., 2017) As this process can result in long-lasting cognitive changes for these patients, finding targets for diagnosis and therapy are of utmost importance.

Diagnostic imaging has been impactful in the assessment of neuroinflammation. Magnetic resonance imaging (MRI) and spectroscopy (MRS) have been integral in investigating suspicious pathologies related to neurological insult. For example, in the analysis of multiple sclerosis (MS), advanced MRI techniques can locate characteristic lesions to guide diagnosis and staging.(Cortese et al., 2019) Positron Emission Tomography (PET) is a more sensitive molecular imaging technique that can non-invasively detect and quantify physiological changes in vivo. PET allows for the determination of pharmacokinetics and distribution of imaging pharmaceuticals and produces three-dimensional (3D) images of the functional processes in the body. As such, many targets are currently being examined to monitor neuroinflammation, and more specifically, microglial activation, at the molecular level using PET.(Chen et al., 2021)

The 18 kDa translocator protein (TSPO), previously known as the benzodiazepine receptor, is found peripherally in organs such as the heart, kidney, lungs, adrenal glands, and others. Lower levels of TSPO are found in the liver and healthy brain. However, this expression is altered and elevated as a result of neuroinflammation via activation of microglia and astrocytes which serve to maintain homeostasis by sensing and responding to the cellular microenvironment.(Chen et al., 2021) Hence, TSPO over-expression has been extensively studied as a biomarker for neuroinflammation and its association with multiple pathologies including Alzheimer’s disease, multiple sclerosis, epilepsy, and stroke.(Werry et al., 2019)

Introduced as a TSPO-binding PET tracer over thirty years ago, [¹¹C]PK11195 has been thoroughly studied and validated in neurological diseases including Alzheimer’s disease, multiple sclerosis, Parkinson’s disease, and many others.(Venneti et al., 2006) Unfortunately, [¹¹C]PK11195 displays poor signal-to-noise and non-specific binding that limits its use in studying the neuroinflammatory response in the CNS. As a result, a second generation of TSPO PET tracers were developed that possess higher signal to background ratios and improved specificity toward TSPO in glial activation and astrocytes in many brain pathologies. One of these tracers, [¹⁸F]DPA-714 has been evaluated in brain assessment of patients experiencing Parkinson’s Disease(Lavisse et al., 2021), Alzheimer’s Disease(Golla et al., 2015), stroke(Lin et al., 2023), epilepsy(Dickstein et al., 2019), depression(Yrondi et al., 2018) and concussion.(Neumann et al., 2023)

The goal of this work was to develop a robust, automated method to produce [¹⁸F]DPA-714 routinely for clinical and preclinical research using an IBA Synthera^® radiosynthesizer with high activity yield and a production time of approximately one hour. To this end, we optimized and validated a radiosynthesis method in accordance with the guidelines of USP <823>.

2. Materials and Methods

2.1. General

All chemicals were purchased from Sigma Chemical Company (St. Louis, MO), unless otherwise noted. All aqueous solutions were prepared with ultrapure water (Milli-Q Integral Water Purification System, Millipore Corp.; 18.2 ΜΩ·cm resistivity) or sterile water for injection. The radiochemical precursor (1) N,N-Diethyl-2-(2-(4-(2-toluenesulfonyloxyethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (>95%), as well as the non-radioactive DPA-714 reference standard, N,N-diethyl-2-(2-(4-(2-fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide (>95%) and tetrabutylammonium hydrogen carbonate, TBA HCO₃, solution (0.075 M in ~9% ethanol and water) were obtained from ABX (Radeberg, Germany) and used as supplied. High purity anhydrous solvents, e.g., dimethyl sulfoxide (>99.8%), acetonitrile (99.8%) and ethanol, (>99.5%, 200 proof, USP grade) were used without further purification.

The [¹⁸F]DPA-714 synthesis method described herein was developed and optimized using an automated Synthera^® radiosynthesizer (Version 1, IBA Molecular) employing an Integrated Fluidic Processor^™ (IFP) cassette. Briefly, the Synthera^® is equipped with a row of rotary actuators to which a disposable IFP^™ cassette can be attached. The cassettes are used as supplied without any modifications and have four vial slots of either 11 mm or 13 mm diameter. For [¹⁸F]DPA-714 synthesis as depicted in Figure 1, slot 1 holds an 11 mm vial containing the phase transfer catalyst/base solution, the precursor solution (13 mm vial) is placed in slot 2, and slot 4 contains a 13 mm vial with the HPLC mobile phase for subsequent purification.

FIGURE 1.

Synthera setup for [¹⁸F]DPA-714 synthesis.

In addition, the Synthera^® was accompanied by an in-house, custom-built semi-preparative HPLC purification system (RICO, Remote Injector and fraction COllector), consisting of a binary HPLC pump (Agilent), a Rheodyne valve-controlled 5 mL sample loop and injection port, and radioactivity detector. The module was further connected to an in-house custom-made solid phase extraction unit (SPE) consisting of several valves, syringe drivers, and reagents for removal of HPLC solvents and final formulation of the tracer.

The HPLC module (RICO), reformulation system (SPE), and final product line were cleaned and sterilized by rinsing tubing with 15 mL sterile water, 15 mL sterile 70% ethanol, and then dried with nitrogen prior to each synthesis run. In addition, a new, sterile IFP^™ cassette was used for each production. All radioactive work was accomplished in a Comecer BBS2 hot cell. The ¹⁸F-labeled materials were measured using a Capintec CRC-15PET dose calibrator. Analytical HPLC was performed on a 1200 Series Agilent LC system using both diode array detection and a Bioscan Flow-Count radionuclide detector (Eckert & Ziegler - Bioscan). Volatile organic impurities analysis by gas chromatography was carried out using an Agilent model 8890 GC system. Both the LC and GC equipment were controlled by Agilent’s OpenLab CDS ChemStation software.

2.2. Radiosynthesis of [¹⁸F]DPA-714

Briefly, [¹⁸F]DPA-714 was produced by fluorination of a tosyl-functionalized precursor based on syntheses reported by Damont et al.(Damont et al., 2008) and Kuhnast et al.(Kuhnast et al., 2012) (See Figure 3.)

FIGURE 3.

Synthesis scheme of [¹⁸F]DPA-714 as produced on the IBA Synthera

Aqueous [¹⁸F]fluoride, produced by the ¹⁸O(p,n)¹⁸F nuclear reaction in an IBA Cyclone^® 18/9 cyclotron from ¹⁸O-enriched water, was transferred from the target to a v-vial in a dose calibrator within the hot cell using argon flow (99.9999% high purity – Nexair). After measuring the radioactivity value, the [¹⁸F]fluoride solution was passed through a QMA cartridge (Myja Scientific - pretreated with 2 mL of 1 M sodium bicarbonate and rinsed with 5 mL water) to capture the [¹⁸F]fluoride and separate it from the [¹⁸O]water. [¹⁸F]fluoride was then eluted from the QMA cartridge into the reactor vial with a 650 μL solution of 2 mg TBA HCO₃ (90 μL 0.075M stock solution + 560 μL acetonitrile). This fluoride/TBA HCO₃ solution was then dried under reduced pressure (30 kPa) with argon flow at 110°C for 300 seconds to remove the acetonitrile and water. Anhydrous dimethyl sulfoxide (0.7 mL) containing 2 ± 0.2 mg of the tosyl precursor (1) was added to the reactor vessel. The reactor was sealed and heated at 165°C for 5 minutes. The reaction mixture was cooled to room temperature, and the solution was then diluted for HPLC purification by the addition of 4 mL of HPLC eluent (60% 0.1M ammonium acetate, pH 10 / 40% acetonitrile).

2.3. Purification and Reformulation

Using the custom-built RICO unit, the crude reaction mixture was purified on a Phenomenex Gemini C18 column (250 × 10 mm, 5 μm, 110 Å) with a mobile phase consisting of 60% 0.1M ammonium acetate, pH 10/40% acetonitrile at a flow rate of 5 mL/min at room temperature. The final product was manually collected between 22–25 minutes. Unreacted [¹⁸F]fluoride eluted at approximately 3–4 minutes.

Using the custom-built SPE system, the purified fraction was collected into a vial containing 20 mL of water to dilute the product and reduce the concentration of acetonitrile. This diluted solution containing purified [¹⁸F]DPA-714 was loaded onto a Sep-Pak tC18 Plus Short cartridge (preconditioned with 3 mL ethanol followed by 10 mL water – Waters Corp; Milford, MA) and the eluant sent to waste. The cartridge was then washed with 10 mL of water followed by elution with 0.6 mL of ethanol into a reformulation vial followed by 15 mL of sterile saline. The final product was transferred to a sterile, vented vial fitted with a 0.22 μm sterilizing filter (PVDF, 33 mm; Millipore).

2.4. Quality Control Testing

Several quality control tests were performed on the [¹⁸F]DPA-714 product, prior to release, to assure the quality of the final dose in accordance with guidelines from USP <823> Radiopharmaceuticals for Positron Emission Tomography-Compounding. The final product was confirmed to be clear and colorless with no visual evidence of cloudiness or particulate matter as per USP <823> and USP <631> Color and Achromicity.

2.4.1. HPLC for identification, chemical and radiochemical purity, and molar activity determination

The [¹⁸F]DPA-714 final product [at least 1.11 MBq (30 μCi)] was injected onto an Agilent Zorbax Eclipse XDB-C18 analytical column (150 × 4.6 mm, 5 μm) for analysis. An isocratic mobile phase containing a 50:50 mixture of ultrapure water and acetonitrile with a flow rate of 1.0 mL/min was used to elute the product with UV (265 nm) and radioactive detection. A [¹⁹F]DPA-714 stock solution (0.2 mg/mL in acetonitrile) was used as a standard for [¹⁸F]DPA-714 identification by retention time comparison. The radiochemical purity of [¹⁸F]DPA-714, expressed as a percent, was determined from the peak area of [¹⁸F]DPA-714 and the total peak areas in the chromatogram as detected by a radioactivity detector. The [¹⁸F]DPA-714 final dose was analyzed in duplicate, in addition to a 1:1 (vol) co-injection with the reference standard.

For molar activity determination, the HPLC sample vial containing the final dose of [¹⁸F]DPA-714 was measured for radioactivity. The mass of DPA-714 in the sample was measured by UV absorbance at 265 nm and quantified by measuring the peak area in comparison to the reference standard. Using these values, along with the molecular weight of DPA-714 (398.47 mg/mmol), the molar activity was calculated in Bq/mmol and decay corrected to expiration time.

2.4.2. GC for volatile organic impurities analysis

In order to determine the amount of volatile organic impurities/residual solvents in the final sample, a small aliquot of the [¹⁸F]DPA-714 (0.5 μL) was analyzed by GC using a DB-WAX column (J & W 122–7032, 30 m × 250 μm × 0.25 μm). The oven temperature was set to 60°C for 1 minute, then ramped to 140°C at a rate of 60°C/min and held for 2 minutes, then ramped to 195°C at 100°C/min and held for 1 minute. The total run time was 5.9 minutes, and the front inlet heater was set to 200°C. The split ratio was 15:1, hydrogen flow was 40 mL/min, and the air flow was 400 mL/min. The GC peak retention times and areas were compared to standards of ethanol (1.0%; RT ~ 1.61 min), acetonitrile (0.03%; RT ~ 1.83 min), and DMSO (0.03%; RT ~ 4.32 min).

2.4.3. Other QC testing

In addition, the pH of the final formulation of [¹⁸F]DPA-714 was tested by spotting on a narrow range pH indicator strip and the color was compared to the color range chart provided by the manufacturer. The radionuclidic identity was determined on the final product by observing the radioactive half-life (t_½) using a Capintec CRC-15PET dose calibrator with measurements taken at 10 minute intervals over a period of 20 min and the half-life was determined. Radionuclidic purity was determined on a sample of the final product using a MUCHA multi-channel analyzer and Gina software (Elysia-Raytest) using 10 min sample counting times, spaced at 10 minutes, with a total of 4 measurements. The resultant gamma spectrum was analyzed for the presence of identifiable photopeaks which are not characteristic of F-18 emissions and 99.5% of the observed gamma emissions should correspond to the 0.511 MeV, 1.022 MeV, or Compton scatter peaks of F-18. These results were confirmed by analysis on a high-purity germanium detector connected to a spectrum analyzer (Canberra MCA). The final drug product was tested for the presence of bacterial endotoxins utilizing the Endosafe^®-PTS^™ unit (Charles River) to determine the endotoxin concentration in a sample. Sterility was tested using the direct inoculation method as recommended by the USP, and the samples were observed over 14 days.

Residual TBA HCO₃ was determined by visual spot test on silica by over-spotting each sample with a MeOH/NH₄OH solution and developing in an iodine chamber by adapting the method of Kuntzsch, et al.(Kuntzsch et al., 2014) Reference standards of 0, 96, 192, and 250 μg/mL TBA HCO₃ were prepared in a solution of HPLC purification eluent. Each standard was spotted (1 μL) on a glass-backed silica TLC plate. After drying, 10 μL of methanol/25% ammonium hydroxide solution (9:1) was applied on top of each spot. The plate was then placed in an iodine chamber to develop for one minute. Samples were visually compared to reference standards for interpolation of TBA HCO₃ concentration.

3. Results and Discussion

[¹⁸F]DPA-714 has been extensively studied in the context of TSPO expression and CNS disease, thus, a significant amount of research effort has been devoted to ensuring researchers have access to this tracer. As such, the synthesis of [¹⁸F]DPA-714 has been automated on several synthesis platforms such as the GE TRACERlab^™ FX FN (Kuhnast et al., 2012), Trasis AllInOne (Cybulska et al., 2021), and more recently the Sofie Elixys Flex/Chem (McCauley et al., 2022).

The goal of this study was to optimize the synthesis of [¹⁸F]DPA-714 on an automated Integrated Fluidic Processor^™ (IFP)-based Synthera^® synthesis module from IBA Molecular, which, to our knowledge, has not been reported to-date. The Synthera^® was accompanied by two custom-built purification units, RICO and SPE, made in-house to convert it into an all-in-one synthesis and purification system that can deliver final formulations at the end of every production run without the need for manual intervention.

We based our synthesis protocol on previously published methods (Damont et al., 2008; Kuhnast et al., 2012) with some modifications. Figure 4 shows a schematic representation of the steps in our radiosynthesis, purification and final formulation of [¹⁸F]DPA-714.

FIGURE 4.

Process flowchart for synthesis of [¹⁸F]DPA-714

We opted to move forward with DMSO as the solvent and reduced the mass of precursor to 2 mg per synthesis. The starting radioactivity was calculated from the difference between transferred activity from the cyclotron as measured in the v-vial and activity remaining in the v-vial post transfer of the [¹⁸F]fluoride to the QMA cartridge. One significant modification to the method is the use of tetrabutylammonium bicarbonate (TBA HCO₃) in the place of Kryptofix^® 222 as a less toxic, yet effective alternative phase transfer catalyst. The phase transfer catalyst/base solution contained a mixture of 90 μL TBA HCO₃ ethanolic solution mixed with 560 μL acetonitrile, resulting in a TBA HCO₃ concentration of 2 mg in 650 μL. The reaction temperature and time were set at 165°C and 5 min, respectively. The duration of the entire synthesis and purification was approximately 65 minutes. [¹⁸F]DPA-714 was typically produced with an EOS activity yield of 24.6 ± 3.8% and a molar activity of > 18.5 TBq/mmol (500 Ci/mmol), which is in good agreement with synthesis yields reported elsewhere (TABLE 1).

TABLE 1.

Comparison of reported syntheses of [¹⁸F]DPA-714 on various radiosynthesizers

Parameter	GE TRACERlab FX FN (Kuhnast et al., 2012)	Trasis AllInOne (Cybulska et al., 2021)	Sofie Elixys Flex/Chem (McCauley et al., 2022)	IBA Synthera V1 (this work)
Phase Transfer Agent	Kryptofix® 2.2.2	TEAB	Kryptofix® 2.2.2	TBA HCO3
Mass of Precursor	4–5 mg	4 mg	6 mg	2 mg
Reaction Solvent	DMSO	Acetonitrile	Acetonitrile	DMSO
Reaction Temperature	165 °C	90 °C	100 °C	165 °C
Reaction Time	5 min	10 min	15 min	5 min
Reported Yield @ EOS	30 – 35% (n > 30)	45.7 ± 3.21% (n = 3)	13.5 ± 3.04% (n = 3)	24.6 ± 3.8% (n = 5)
Molar Activity in GBq/μmol (Ci/μmol)	74 – 222 (2 – 6)	166.5 ± 48.1 (4.5 ± 1.3; n = 3)	136.9 ± 51.8 (3.7 ± 1.4; n = 3)	140.6 ± 25.9 (3.8 ± 0.7; n = 3)
Total Synthesis Time	50 min	62–64 min	90 min	65 min

Routinely, [¹⁸F]DPA-714 was produced with > 99.9% radiochemical purity. Three process validation syntheses were conducted to qualify the radiosynthesis for clinical research production and IND submission (Table 2). Analysis of representative samples with a high purity germanium detector confirmed > 99.5% radionuclidic purity of fluorine-18.

TABLE 2.

Quality control results from process validation productions of [¹⁸F]DPA-714

QC Test	SPECIFICATION	BATCH 1	BATCH 2	BATCH 3
Initial Synthesis Activity	N/A	7.93 GBq (214.4 mCi)	7.93 GBq (214.4 mCi)	7.86 GBq (212.5 mCi)
Visual Appearance	Clear and Colorless	Clear and Colorless	Clear and Colorless	Clear and Colorless
pH	4.5 – 7.5	5.5	5.5	5.5
Radiochemical Purity	≥ 95% [18F]DPA-714	100%	100%	100%
Radionuclidic Identity	Observed t½ is 105 to 115 minutes	110.2 min	111.1 min	109.6 min
Radionuclidic Purity	≥ 99.5% 18F	99.77%	99.80%	99.79%
Mass Limit	DPA-714: ≤ 9 μg/dose	0.51 μg/dose	0.65 μg/dose	1.23 μg/dose
Molar Activity	≥ 18.5 TBq/mmol (500 Ci/mmol) at TOIa	37.2 TBq/mmol (1005 Ci/mmol) at expirationb	28.0 TBq/mmol (756 Ci/mmol) at expiration	26.3 TBq/mmol (711 Ci/mmol) at expiration
Volatile Organic Impurities	DMSO ≤ 0.5%	< 0.001%	< 0.001%	< 0.001%
	Acetonitrile ≤ 0.041%	0.011%	0.004%	0.007%
Bacterial Endotoxin (LAL)	≤ 175 EU/dose	< 2.5 EU/mL	< 2.5 EU/mL	< 2.5 EU/mL
Filter Integrity	≥ 50 psi breaking pressure	52.6 psi	53.2 psi	55.4 psi
Sterility	no growth observed over 14 days	No Growth	No Growth	No Growth
Ethanol Content (Not a release criterion)	≤ 5%	3.44%	3.46%	3.01%

^atime of injection

^bexpiration set to 4 hours based on molar activity during stability validation

TBA HCO₃ has been used successfully as a phase transfer catalyst in the production of ¹⁸F-radiopharmaceuticals in our facility and by others.(Vavere et al., 2018; Wagner et al., 2009) There are no reported toxicities for this compound or several other tetrabutylammonium compounds.(April 11, 2009; December 10, 2014; January 19, 2015; May 21, 2013) However, a general report on the toxicology of quaternary ammonium compounds (QACs) outlined in the material safety data sheet (MSDS) of tetrabutylammonium chloride concluded that “from human testing of different QACs the generalized conclusion is obtained that all the compounds investigated to-date exhibit similar toxicological properties.” The same report stated that for some QACs the LD₅₀ value is several hundred times lower by i.p. or i.v. than the oral route, although toxicity can vary between compounds. The routinely used phase transfer catalyst, Kryptofix^® 222, has a reported LD₅₀ listed as > 300 – 2000 mg/kg (oral, rat) and 35 mg/kg (i.v., rat).(Baudot et al., 1977) The United States Pharmacopoeia (USP) does not provide a recommended release limit for TBA HCO₃, however, the European Pharmacopoeia does have an established release limit of 2.6 mg/V (V = injected volume).(2014) Our synthesis intentionally requires only 2.0 mg of TBA HCO₃ such that even if all the reagent were to be carried through to the final product, this recommended release limit would not be reached. The majority of the TBA HCO₃ will be purified out of the final product via semi-preparative HPLC during the final purification. Residual TBA HCO₃ in [¹⁸F]DPA-714 preparations (n = 5) was determined by visual spot test by comparison to a range of reference standard concentrations.(Kuntzsch et al., 2014) Results showed all samples consistently contained less than 96 μg/mL (see Figure 5 for example), confirming the concentration of TBA HCO₃ remaining in the final product is significantly below the recommended release limit established by the European Pharmacopoeia. These results negate the need for routine testing of TBA HCO₃ levels in our final formulation.

FIGURE 5.

Example color spot test for residual TBA HCO₃ in [¹⁸F]DPA-714 product.

It is common for FDA-approved and research PET tracers to contain ≤ 10% ethanol as an excipient in the final solution. Serdons et al. examined the question of the presence of ethanol in radiopharmaceutical injections, discussing the potential side effects.(Serdons et al., 2008) They presented that solutions containing ethanol should be diluted in saline to maintain the injection as isotonic and prevent hemolysis, whereas dilution of blood in an ethanol-water (7:93, v/v) mixture can induce hemolysis. While they mentioned that even a generally acceptable 20 mL injection of 10% ethanol solution in an adult would result in a temporary and rapidly decreasing blood alcohol below most limits for driving, special attention should be paid to injections for children. The World Health Organization has released guidance on the development of pediatric medicines and points to consider in pharmaceutical development, stating that as a solvent or co-solvent “For the paediatric population the maximum of ethanol is: 0.5% for children under 6 years, 5% for children 6–12 years, and 10% for children over 12 years (Kristensen, 2008)”. Based on this guidance, our synthesis was designed with a preparation of 3.8% ethanol in saline to remain isotonic and less than the 5% recommendation.

4. Conclusion

In summary, an automated method for the radiochemical synthesis of [¹⁸F]DPA-714 was developed and optimized on a commercially available IBA Synthera^® radiosynthesizer equipped with a custom-built purification and reformulation unit. We modified precursor mass, substituted a less toxic phase transfer catalyst, and reduced ethanol concentration resulting in improved synthesis and reformulation methods suitable for clinical research production in the pediatric setting. This method allows reliable production of [¹⁸F]DPA-714 with 24.6 + 3.8% radiochemical yield (EOS), high radiochemical purity (> 99.9%), and molar activity [> 18.5 TBq/mmol (500 Ci/mmol)]. Finally, the synthesis was process-validated with comprehensive quality control testing in accordance with USP <823>, qualifying it for clinical research applications.

FIGURE 2.

Schematic of RICO (Remote Injector fraction COllector) and SPE (Solid Phase Extraction) custom, in-house modules.

Highlights:

• An IND-approved method for production of [¹⁸F]DPA-714 on an IBA Synthera^® is reported.

• Reduction of precursor mass and replacement of Kryptofix^® 222 as phase transfer agent support production of tracer in reliable quantities for clinical research.

• Justification of 5% injected ethanol in pediatric PET tracer preparations was outlined.