Development and Optimization of ¹¹C-Labeled Radiotracers: A Review of the Modern Quality Control Design Process

Abstract

Introduction - Several ¹¹C-tracers have demonstrated high potential in early diagnostic PET imaging applications of neurodegenerative diseases including Alzheimer’s and Parkinson’s disease. These radiotracers often track critical biomarkers in disease pathogenesis such as tau fibrils ([¹¹C]PBB3) or β-amyloid plaques ([¹¹C]PiB) associated with such diseases. Purpose - The short review aims to serve as a guideline in the future development of radiotracers for students, postdocs and/or new radiochemists who will be synthesizing clinical grade or novel research ¹¹C-tracers, including knowledge of regulatory requirements. We aim to bridge the gap between novel and established ¹¹C-tracer quality control (QC) processes through exploring the design process and regulatory requirements for ¹¹C-pharmaceuticals. Methods - A literature survey was undertaken to identify articles with a detailed description of the QC methodology and characterization for each of the sections of the review. Overview - First a general summary of ¹¹C-tracer production was presented; this was used to establish possible places for contamination or assurances for a sterile final product. The key mandated QC analyses for clinical use were then discussed. Further, we assessed the QC methods used for established ¹¹C-tracers and then reviewed the routine QC tests for preclinical translational and validation studies. Therefore, both mandated QC methods for clinical and preclinical animal studies were reviewed. Last, some examples of optimization and automation were reviewed, and implications of the QC practices associated with such procedures were considered. Conclusion - All of the common QC parameters associated with ¹¹C-tracers under clinical and preclinical settings (along with a few exceptions) were discussed in detail. While it is important to establish standard, peer-reviewed QC testing protocols for a novel ¹¹C-tracer entering the clinical umbrella, equal importance is needed on preclinical applications to address credibility and repeatability for the study.

1. Background

Positron emission tomography (PET) is a highly selective 3D imaging technique enabling real-time, in vivo, quantitative visualization of biological processes.^1,2 As a research tool, PET reveals the mechanisms of disease initiation and progression and the biological uptake and metabolic fate of newly developed therapies or drugs in real time.¹ PET can also be used to observe the pharmacokinetic properties of therapeutic compounds, drugs, or substances of abuse in clinical and preclinical studies.^1,3

Since radiotracers for PET scans are administered parenterally to human subjects or patients, quality control (QC) guidelines are thorough and must be adhered to strictly to ensure a safe procedure can be conducted.^1,4,5 Several regulatory bodies enforce them: the US Federal Drug Administration’s Code of Federal Regulations (CFR) Title 21, part 601, subpart D, diagnostic radiopharmaceuticals, stipulates precautions and safety measures;^1,5 the European Medicines Agency (EMA) refers to European Pharmacopeia (EP) chapter 5, a general monograph on radiopharmaceutical preparations;⁶ and the USP general chapter 823 addresses radiotracers.⁴ These regulations must be considered during radiotracer development for eventual clinical use. The choice of reagents and time constraints of excessive purification, if harmful compounds are employed or produced, should be considered. Therefore, the use of toxic class 1 solvents, or chemicals that would be difficult to detect should be avoided.⁷

Radionuclides used for PET undergo spontaneous positron emitting β⁺ decay, with resulting short half-lives (T_1/2), examples include: oxygen-15 = 2.03 min, copper-62 = 9.67 min, nitrogen-13 = 9.96 min, carbon-11 = 20.4 min, gallium-68 = 67.7 min, and fluorine-18 = 109.8 min.^1,4,5 The emitted positrons collide with electrons and annihilate; two directly opposed γ rays, each with 511 keV, are subsequently detected by a ring in the PET scanner.¹ The copopulated decay events computationally form a 3D rendered radiotracer distributionin vivo that can be overlaid with a CT scan.¹

Use of ¹¹C-labeled compounds as PET tracers is popular as the labeled compound is identical to the original biomolecule both in terms of its biologic function and structure, without having to change a functional group or chelating a metal to radio-label the desired compound,⁸ and it is currently the second most used radionuclide, with fluorine-18 being the most common.^9,10 An important factor for the popularity of carbon-11 (¹¹C) as a radionuclide is its half-life, which is sufficiently long to perform multistep syntheses and, at the same time, short enough to allow consecutive studies on the same individual in a day. Another factor is the abundant availability of literature in organic chemistry where carbon–carbon bond formation has been extensively researched and can be applied to radiochemistry.^10,11

The short T_1/2 of the radionuclides used in a radiotracer create physical time constraints for the production and QC of such as a radiotracer and means the process must be completed just prior to imaging.¹ To compensate for this limitation, USP 823 provides certain exemptions in the QC process of radiopharmaceuticals compared with other less time sensitive pharmaceuticals: Not all QC testing has to be completed before the radiotracer is used, provided documented validation runs are completed.⁴

The reliable pharmaceutical production of short-lived ¹¹C-compounds require high yields, short reaction times, and efficient purification methods that align with robust, reliable, and optimized QC methods.^9,12

Although there are many recent and ongoing advances in ¹¹C-chemistry, very few radiotracers are pushed through to clinical practice. This short review article will focus on ¹¹C-tracer QC design as this is of great importance for clinical application or extended studies using ¹¹C-tracers. This article briefly reviews the development or optimization of pharmaceutical ¹¹C-tracers for PET imaging, considering the QC and regulatory challenges of both clinical and preclinical studies. The general steps of radiopharmaceutical production that may affect the final QC assessment, either as a potential source of contamination or assurance that the radiotracer is of clinical grade, were assessed.

2. Overview of the General Production and QC Process for ¹¹C-Labeled Radiotracers

Radiopharmaceutical production needs specialized equipment such as a cyclotron, hot cell, radiochemistry module with preparative HPLC, radiation detectors, and a sterile ISO 5 environment.¹³ According to Title 21 CFR part 21 and USP 823, facilities, equipment, personnel, and document procedures must ensure a sterile PET product, although its sterility can only be confirmed through inoculated and incubated growth media after it has been used.^4,14 The chemical synthesis of a parenterally administered radiotracer may take place in an open or closed system, so that some steps may be performed on a benchtop; however, all activities downstream of the membrane filtration should be conducted in a closed, sterile system to minimize contamination risk.⁴ Radiopharmaceutical filtration and dispensing must be performed in an environment with at least an ISO 5 rating.^4,8Figure 1 displays the production and QC steps necessary for compliance with the current regulations. However, some details were omitted, such as several people involved behind the scenes to keep the PET site operational and to establish and maintain current good manufacturing practice (cGMP) compliance.¹⁵

Figure 1.

Flowchart shows the main steps involved in the production and use of a radiopharmaceutical in a clinical setting. Some potential contaminant introducing steps are indicated with red arrows, whereas purification steps are indicated in green arrows, it should be noted contamination sources are highly site and procedure specific.

The production of a ¹¹C-labeled radiopharmaceutical product starts with the cyclotron proton bombardment of nitrogen gas. In the subsequent ¹⁴N(p,α)¹¹C nuclear reaction, a short-lived oxygen-15 (122 s T_1/2) decays to the desired ¹¹C and a byproduct, helium-4.^8,13,16,17 The radio decay process of ¹¹C, to stable boron-11, releases a positron that collides with electrons and annihilate; resulting in two directly opposed (straight angle) γ rays, each with 511 keV.¹⁸ The radionuclide identity and purity of the produced ¹¹C needs to be confirmed in QC processes of the final product.⁴ Periodically the radionuclide purity of decayed retention samples of the ¹¹C-labeled radiopharmaceutical preparations should be tested to confirm the absence of long-lived radionuclides that are produced in the target system of the cyclotron.⁴

The next step (Figure 1) is the synthesis of the chosen ¹¹C-synthon. The cyclotron can produce either [¹¹C]CO₂ or [¹¹C]CH₃ based on the amount of O₂ (0.5–1%) or H₂ (5–10%) respectively introduced into the bombardment cell. Oxygen, and other radio impurities, such as the helium-4 byproduct, is removed using a cryogenic trap (liquid nitrogen) or molecular sieves.⁸ Hydrogen can be removed by trapping [¹¹C]CH₄ in a Porapak column cooled with liquid nitrogen.¹⁰ The literature reports many other ¹¹C-synthons:[¹¹C]CH₃OH, [¹¹C]CH₃I, [¹¹C]CH₃OTf, [¹¹C]CHF₃, [¹¹C]CCl₄, [¹¹C]COCl₂, [¹¹C]HCN and [¹¹C]CS₂.^10,17 Automated methods for gas phase production of routinely used ¹¹C-synthons have been around for over two decades.¹⁹ Methylation by [¹¹C]CH₃I or [¹¹C]CH₃OTf represents the most popular ¹¹C-labeling strategy for the synthesis of ¹¹C-labeled radiopharmaceuticals.¹⁶ The use of [¹¹C]CO₂ directly from the cyclotron has been referred to as the “renaissance” of radiochemistry and is an attractive alternative as it cuts out the yield consuming step of synthon preparation.^10,16,20 However, due to the low chemical reactivity of CO₂, high pressure and temperatures under catalysts are required to achieve the reaction.¹⁰ The use of metal containing catalysts to achieve radiolabeling with [¹¹C]CO₂ does introduce further considerations for purification and subsequent QC of radiotracer²¹ and the use of less harsh radiolabeling conditions and less hazardous catalysts are beneficial.¹⁶ For a more extensive review on ¹¹C-compound synthesis refer to Dahl et al.¹⁰ Both chemical purity and radiochemical purity of the final ¹¹C-compound needs to be assessed prior to clinical use,⁴ the use of an impure synthon might be detrimental to the purity of the final radiotracer.

Further steps are also required to extract moisture from the chosen ¹¹C-synthon to ensure reproducibility and high yields in the radiolabeling reactions to follow.¹⁷ For QC, the data must confirm that the desired radionuclide produced meets the purity standard. Half-life calculations²² and/or gamma-ray spectrometry, such as a multichannel analyzer, can confirm radionuclide identity.^2,4,8,16,23 The EP monograph on radiopharmaceutical preparations requires both half-life determination and gamma-ray spectrometry for radionuclide purity analysis.²³

Radiochemistry happens in a remotely operated hot cell containing an auto synthesis module (Figure 1). Radiolabeling must be optimized to produce high yields of ¹¹C-labeled radiotracer in a short time span.^8,19 Many different ligands, such as sugars, amino acids, peptides, and small molecules have been ¹¹C-labeled to obtain a desired radiotracer.⁸ Commercially available precursors can be used for routine production and has been optimized to react readily with the applicable ¹¹C-synthon.^8,16 Therefore, using certified cGMP compliant precursors is ideal from a QC perspective. However, for the development of a novel radiotracer, a precursor might be produced in-house, which can present challenges and complications for both production and QC steps of the ¹¹C-tracer preparation procedure.

After radio labeling, the radiotracer is isolated from the reaction mixture that contains unlabeled precursor and other harmful compounds.¹⁹ The primary method is preparative high-performance liquid chromatography (HPLC), often in combination with solid phase extraction (SPE),^16,19,23 however, liquid–liquid extraction has also been used.¹² Time is the major challenge for the purification methodology employed.^12,16,19 In addition, bioactivity is often stereospecific, l and d isomers may have to be separated from one another using a chiral selective column.²⁴ This preparative HPLC method can introduce unwanted solvents necessitating residual solvent concentration assessment during QC. Overall, QC methods are deployed to detect any precursor, contaminants, or byproducts remaining in the final product.

The formulation after purification (Figure 1) of the radiotracer usually involves SPE, to remove the mobile phase employed during HPLC purification and to dilute the ¹¹C-compound into its final delivery solution.^1,16,25 Rotatory vacuum evaporation can also be used to remove the mobile phase.^1,19 Usually, saline is employed for injectable radiotracers,^19,26 however a sodium bicarbonate solution can also be used.²⁴ Inhalable [¹¹C]nicotine has been formulated as e-liquid, used in vapes.³ It is imperative that the QC methods be adjusted to accommodate the post formulation sample matrix. Formulation through SPE can also act as a further purification step to remove solvents introduced during preparative HPLC.

The filtration step removes any bacterial contamination and is an assurance for the sterility of the final product if the sterile vial assembly was completed aseptically within an ISO 5 environment. USP 823 mandates the use of a 0.22 μm filter for all PET drugs administered parenterally.⁴ Aseptic Techniques must be used to keep the final product sterile down stream of this filtration step, inside the aseptic workstation.^4,5 During QC, the bubble-point test (Figure 1), is conducted to verify the filter membrane integrity, indicating the membrane filter was not faulty and did not rupture during the filtration step.

During organoleptic analysis, or visual inspection, the production chemist checks the final product for particulate impurities and assess the clarity and color of the solution, ensuring this is consistent with previous batches.^23,27 USP 823 lists the organoleptic test as a release criterion for clinical radiotracers.⁴ The visual inspection should be done before dispensing or taking the QC sample to avoid downstream contamination or removing any potential particulate matter from the vial before the inspection.

Dispensing, the last step in the hot lab before the tracer is used (Figure 1), assesses the yield of the reaction and ensures that enough radiotracer was produced to perform the eventual scan. For a series of scans, a single batch can be divided into sub doses using an ionization chamber dose calibrator.^4,25,28 During QC sampling and transport of the final dispensed dose to the subject, adequate aseptic operations and procedures should be employed to maintain the sterility and integrity of the final ¹¹C-product.⁴ The QC sample should be large enough to facilitate a retention sample (if required) and is representative of the entire batch. It should be noted that normally it is not necessary to retain reserve samples of a ¹¹C-labeled PET radiopharmaceutical batch.⁴ A retention sample is required in case of an out of specification (OOS) investigation, that would involve retesting, or in the case of a conditionally released batch.⁴

Sterility testing of the final product is usually preformed through direct inoculation of growth media with the final product, in conjunction with bacterial endotoxin (BET) testing.⁴ The final sterility testing results are only obtained after the radiotracer has been used, as the broths need to be incubated for several days. The touch plates, as indicated in Figure 1, also refer to air sampling plates to confirm the ISO 5 environment was maintained during final product dispensing and sampling. Air particle counts are also required to demonstrate that the air quality in the environment meets at least IOS 5 standards.

The QC sample should be taken from the final product vial utilizing adequately aseptic operations and procedures to ensure a sterile parenteral PET drug.⁴ For a PET drug produced in sub-batches, appropriate QC must be performed to ensure that each sub-batch conforms to specifications.¹⁴ The sample is subsequently divided (Figure 1) for the required tests. Radiochemical and chemical purity must be assessed using either a radio thin layer chromatography (radio-TLC) scanner or high-performance liquid chromatography (HPLC) with a radiometric detector.^4,7 Radiotracer pH must fall within an acceptable range, depending on the mode of administration, and is determined using either pH strips²⁷ or a calibrated pH meter. The molar activity should pass a set limit to ensure the safe administration of the ¹¹C-tracer.⁴

The decision to release the final radioactive product falls to a person bearing quality assurance responsibilities, such as a principal investigator (PI) or responsible pharmacist (RP), and depends on both the production and QC reports.⁹ Title 21 CFR part 212 outlines a final release procedure that should be followed in cGMP facilities in the United States.¹⁴

The actual PET scan and dose injection is usually performed by a nuclear medical technologist, who must receive confirmation from the authorized quality assurance personnel that the ¹¹C-tracer has been released for use.

2.1. General QC Steps for Pharmaceutical and Radioactive Testing

USP 823 requires documented validation runs of a new product to include evaluation of radiochemical, radionuclide and stereochemical (if applicable) identity and purity.⁴ Sterility and bacterial endotoxin tests (BETs) are required for parenteral PET drugs only.⁴ Any toxic chemical that was used in synthesis or purification steps of the product also needs to be assessed.⁴

Every batch of radiopharmaceutical requires 9 QC tests before it can be administered to human subjects or patients. They address the following: 1) appearance, 2) pH, 3) radiochemical purity, 4) radionuclide identity, 5) concentration, 6) specific activity for drugs with mass-dependent toxicity, 7) residual solvent, 8) chemical purity, and 9) stabilizers.⁴ Every clinically applied ¹¹C-tracer must have acceptance parameters and specifications documented that must be met, prior to its use.^13,14

QC tests can be categorized into two categories: pharmaceutical and radioactive. Pharmaceutical tests ensure molecular identity and physiological compatibility and that any microbial or chemical contamination is within acceptable limits. Radioactive testing ensures that the correct dose is administered and no radiochemical or radionuclide contaminants will interfere with the biodistribution of the radiotracer.^1,8 Radioactive concentration or strength of the final product must also be determined.^1,4

A typical timeline for a ¹¹C-labeled radiopharmaceutical production⁹ and an optimized QC timeline, where the longest analysis (the BET in this case) is started first, followed by the other analysis required before the radiotracer can be released and used¹³ is shown in Figure 2. The time of the associated QC tests vary from ∼15 min for the BET to as little as ∼30 s for pH measurements. The color coding of Figures 1 and 2 indicates the location for each step. In order to optimize time usage, the QC report is compiled as shorter tests are completed. Optimizing the chromatography methods can dramatically shorten the time required to complete the QC process resulting in an increase to the overall yield and molar activity (MA) at the time of administration.⁹ Not all of the QC tests need to be completed before the radiotracer is administered provided that documented validation runs are completed and written procedures are in place.⁴

Figure 2.

Typical production timeline with a staggered QC approach depicted.

The preparation time, as indicated in Figure 2, includes mandated day-of-use checks on the synthesis equipment and system suitability tests for the QC equipment. Day-of-use checks are procedures designed to check key parameters such as the gas supply, vacuum, temperature, and pressure set points at the beginning of each operational cycle. System suitability tests should ensure that the equipment, components, and personnel (i.e., the system) functions correctly to execute the desired analytical test.⁴ Dose calibrators require calibrated radioactive sources, used as reference standards during the day-of-use checks for accuracy and constancy prior to radionuclide purity assessment.¹⁵

2.1.1. HPLC Use in Both Pharmaceutical and Radioactive Tests

The produced radiotracer’s identity and its radiochemical and chemical purity can be assessed by HPLC coupled with a radiodetector and UV detector.^16,23 Radiochemical purity refers to the absence of other radiochemical compounds, whereas chemical purity refers to the absence of other chemical compounds.²⁹ HPLC has been known to be the golden standard for ¹¹C-labeled radiopharmaceutical QC for five decades.³⁰ Conjugation, usually caused by aromatic rings, is usually responsible for a compound’s UV absorption. If a radiotracer does not have a conjugated system, then a different detector will be needed; for example, [¹¹C]choline QC was analyzed with a HPLC with a conductivity detector² or [¹¹C]ER176 employed a ESI MS.²⁵ Laboratories designing novel radiotracers may have to acquire a detector for QC.

The concentration of the radiotracer needs to be determined and is reported as the molar activity (MA), as reported in the SI unit GBq/μmol. The MA is cited in both the European Pharmacopoeia (EP, chapter 5) and USP 823 as a release criteria for radiopharmaceuticals.^4,6

Tailing factor and resolution, sometimes referred to as column efficiency, should be assessed daily as part of the day-of-use checks.^4,5 To confirm consistency, all QC methods should be replicated daily and the variance in peak area, or calculated concentration, for at least two standard injections determined.⁴ Internal or external reference standards, with a known concentration, are required for these HPLC day-of-use checks. Reference standards should be prepared from well-characterized or certified materials.⁴

HPLC is commonly used to determine the concentration of the cold precursor and chemical impurities before a radiotracer is used. For neurological receptors, the precursor concentration is important as there are a minimal number of binding sites present, it is less important for oncological applications.⁸

Cold carbon-12 standards for calibration curves are generally commercially available for routinely produced radiopharmaceuticals.

2.1.2. GC Use in Radiopharmaceutical QC

Gas chromatography (GC) is routinely employed for quantification of volatile compounds that might include reagents, solvents, catalyst, or precursor in the final radiotracer formulation.^1,16 USP 467 specifies permissible daily exposure limits for solvents present in peripheral drugs.¹⁶ For literature examples, GC with a flame ionization detector (GC-FID) was used to measure phenylsilane (PhSiH₃) used for direct [¹¹C]CO₂ labeling.¹⁶ Additionally, for [¹¹C]acetate, GC was used to determine the concentration of precursor still present in the final product.² GC should be used to evaluate the final ¹¹C-tracer formulation for any solvent used during the radiotracer synthesis or purification; other potential solvent contaminating steps might be overlooked, such as solvents employed for cleaning equipment or sterilization of the ISO 5 environment.

2.2. Sterility Testing of Radiotracers

Because the sterility test results for the ¹¹C-tracer is only obtained after a suitable incubation period, aseptic procedures need to adequately ensure the sterility of the final product.⁴ The use of sterile empty vials, transfer lines, needles, membrane filters, and other components used for product vial assembly must be established with written specifications for the identity and quality of these components.⁴ The efficiency of the aseptic procedures is initially demonstrated through three validation runs and later through trending of daily testing of produced batches.

2.2.1. Bacterial Endotoxin Test (BET)

The bacterial endotoxin test (BET), which can be completed in approximately ∼15 min, determines the concentration of endotoxins present in the final product through the use of Limulus Amoebocyte Lysate (LAL).^8,23 The FDA has accepted BET tests since 1977, and it can be used as a final product test under specified conditions.³¹ Specific guidelines were published in USP 85 for performing bacterial endotoxin tests.³²

BET is generally employed as a fast-paced proxy for sterility testing that is still in progress when a ¹¹C-tracer is released for human use.

2.2.2. Membrane Filter Integrity

USP 823 mandates the use of a 0.22 μm or finer filter for all PET drugs administered parenterally.⁴ Filter integrity gives a quick indication of the final product’s sterility before plate incubation is completed.⁸ The EP radiopharmaceutical preparation monograph prescribes the bubble-point test: The used 0.22 μm filter is connected to a compressed-air or inert-gas supply and attached to a hypodermic needle dipped in water. The pressure is gradually increased until a continuous stream of bubbles is observed, indicating a rupture in the membrane filter. This maximum pressure, at which the filter ruptured, should meet a specification as set by the manufacturer.^6,23

2.2.3. Sterility Testing through Growth Medium Inoculation

Sterility of the final product is assessed by direct inoculation of an aliquot of the ¹¹C-labeled radiotracer into a growth medium; typically, thioglycolate and tryptic soy broths are used for this purpose. In addition to this, sabouraud dextrose agar plates, both touch and air, are used to demonstrate the sterility of the ISO 5 environment. The broths and plates are incubated at appropriate temperatures (25–35 °C) depending on the supplier specification, typical incubation times are 48 h for plates and 14 days for the broths. The inoculated broths solutions should remain clear after the incubation period, indicating that no live microbes were present.^23,33 Alert and action limits should be established for colony’s observed on the plates, a typical alert level is set at less than three growths per plate.⁴ The sterility testing can be conducted after the ¹¹C has decayed, but must generally be started within 30 h after the completed production.¹⁴ USP 823 mandates the inoculation of both tryptic soy and thioglycollate broths for the first production of a radiotracer every production day,⁴ while USP 71 gives specific guidelines on how the broths should be prepared and incubated.³³

2.3. QC for Preclinical Studies and Considerations for Novel Radiotracers

Although full QC testing is not required, the credibility and repeatability of preclinical studies rely on the efficacy of the performed experiments, thus the identification of byproducts and an evaluation radiochemical purity is needed.³⁴ HPLC is commonly used to identify radioactive intermediates and chemical impurities. Furthermore, HPLC can also be used to assess the reaction yield of the target radiotracer.⁸ Additionally, chiral chromatography can also be used to determine the radiochemical purity of the l and d configurations of a particular radiotracer when a racemic mixture might be produced.⁸

Combining HPLC with mass spectrometry (HPLC-MS) creates a powerful tool for optimizing a novel synthesis procedure to develop a radiotracer. MS can identify byproducts and identify impurities to consider when designing a QC procedure.^23,25 Nuclear magnetic resonance (NMR) can confirm that the target compound was produced and identify byproducts through characterization of the chemical structure.^23,35

Synthesizing an equivalent cold compound allows for the optimization of reaction conditions and identification of intermediates or byproducts using NMR or MS without the time constraints or safety concerns associated with a radioactive compound.^23,25 Procuring certified standard compounds for novel or nonroutinely produced radiotracers used in preclinical studies may be difficult and hamper quantitative analysis.

2.4. Considerations for clinical studies

Analytical methods must be validated before use in radiopharmaceutical QC.³⁶ The production procedure and system for a novel radiotracer or a significant alteration to a synthesis method for an existing tracer must also be validated.^16,23,25 USP 823 mandates a documented validation study of three complete batches.⁴ Stability testing is a requirement for radiotracers and must be assessed before clinical use.^5,8

When a novel radiopharmaceutical or synthesis procedure is being developed that is intended for eventual clinical use there are some advisable steps: 1) Avoid very toxic chemicals or class 1 solvents that are not permissible even at trace levels. 2) Do not use a chemical that would be hard to detect making validation difficult. 3) Use only commercially available high-purity compounds that are supplied with a COA.⁷

Analytical methods need to be validated, demonstrating through documented evidence that a method, process, or system meets its intended requirements and is able to perform the required QC tests accurately.^4,15

The European Pharmacopoeia contains general monographs for a number of analytical tests that describe instrumental conditions, standards, mobile phase, and columns. If a monograph exists for a specific compound to be assessed and is successfully verified, then no validation procedure is required for that analytical method.^6,36

Current good manufacturing practice (cGMP) principles require continued risk assessment through internal and external audits that should identify any possible hazardous contamination that must be addressed through thorough QC.^7,15

It is recommended to not rely solely on UV detection, especially for a radiotracer synthesis that does not employ a preparative HPLC as some toxic compounds may go undetected.³⁷ However, there are not many articles that mention additional QC investigation, such as inductively coupled plasma atomic emission spectroscopy (ICP-AES) to analyze trace levels of heavy metal concentration,³⁸ although metal catalysts are commonly used for ¹¹C-synthon production.

2.4.1. Deviations, Reprocessing, OOS, Conditional Releases, and CAPA

In the inevitable event that production or QC procedures require a deviation from the standard operating procedure (SOP) or an OOS result is obtained, written SOPs for dealing with the situation should be in place.¹⁵ Any QC result that does not meet the established acceptance criteria, is considered OOS, although this does not necessarily mean a batch rejection is required.⁴ A conditional final release might be applicable when a required QC test for a ¹¹C-tracer cannot be completed due to a technical failure of the QC equipment; however,⁴ a conditional release is not possible if radiochemical identity and purity cannot be determined.⁴ A retention sample must be maintained for the conditionally released batch, and the omitted QC test should be done when the malfunction has been resolved if the retest result would still be meaningful.⁴

An ¹¹C-tracer batch might be reprocessed (only by established procedure) if an OOS test result was obtained for either the bubble point, pH, or chemical purity tests. Reprocessing might include a pH adjustment, filtration with another 0.22 μm membrane filter or a second prep-HPLC run.⁴

An investigation SOP needs to be in place for when an OOS result is obtained, a decision tree might be a useful option for personnel to follow in such an event.⁴ All deviations should be documented, both for trending purposes and to ensure corrective and preventative actions (CAPA) will take place.¹⁵

3. Literature Review on Established ¹¹C-Labeled PET Tracers QC Methods

3.1. Chemical and Radiochemical Purity QC

For an ¹¹C-tracer radiochemical purity is defined as the absence of other radiochemical compounds/species, thus a radio-chemically pure sample may contain other nonradioactive chemicals.²⁹ Whereas chemical purity of a ¹¹C-tracer implies the absence of other chemical compounds, a chemically pure sample may contain ¹¹C-labeled materials.²⁹ It should be noted that any quantitative analysis requires a calibration curve and thus a reference standard.¹⁵

Table 1 summarizes the QC methods used to measure radiochemical and chemical purity for some established ¹¹C-labeled radiopharmaceuticals reported in the literature, used for clinical research, or that have been employed for research purposes for a long time. A Google scholar search for ¹¹C-tracer synthesis articles was used to find the references listed in Table 1, and articles without a detailed QC section were excluded.

Table 1. Identification, Radiochemical, and Chemical Purity Methods of Selected ¹¹C-Tracers.

radiotracer	type of purity assessment	chromatography method	detector	solid phase	ref(s)
[11C]acetate	radiochemical	HPLC	UV and radio	basic anion exchange resin	(2,37)
	chemical	GC	MS		(2)
	radiochemical and chemical	HPLC	refractive index, UV, and radio	reverse phase	(2,37)
[11C]choline	radiochemical and chemical	HPLC	conductivity and radio	cation exchange	(2,22)
	radiochemical and chemical	HPLC	refractive index and radio	reverse phase with counterion in mobile phase	(2)
[11C]CPPC	radiochemicaland chemical	HPLC	UV and radio	C18 reverse phase	(39,40)
[11C]DTBZ	radiochemicaland chemical	HPLC	UV and radio	C8 reverse phase	(41)
[11C]ER176	radiochemical and chemical	HPLC	HRMS (ESI)	C18 reverse phase	(25)
[11C]glyburide	radiochemical and chemical	HPLC	UV and radio	C18 reverse phase	(12)
[11C]HC070	radiochemical	HPLC	UV and radio	C18 reverse phase	(26)
l-[11C]lacate	radiochemical and enantiomeric purity	HPLC	UV and radio	chiral column (Cosmosil HILIC)	(24)
[11C]loperamide	radiochemical	HPLC	UV and radio	C18 reverse phase	(42)
[11C]LY2795050	radiochemical, chemical and enantiomeric purity	HPLC	UV and radio	C18 reverse phase in combination with a chiral separation	(43,44)
[11C]methionine	radiochemical, chemical and enantiomeric purity	HPLC	UV and radio	reverse phase with chiral separation	(8,45)
	radiochemical	TLC	radio TLC scanner	silica gel-based plates	(8)
	radiochemical and chemical	HPLC	UV and radio	reverse phase with chiral separation	(2)
	radiochemical and enantiomeric purity	TLC	radio TLC scanner	TLC with chiral separation	(2)
[11C]MPC-6827	radiochemical and chemical	HPLC	UV and radio	C18 reverse phase	(35,46,47)
[11C]ORM-13070	radiochemical	HPLC	UV and radio	C18 reverse phase	(48,49)
[11C]PiB	radiochemical and chemical	HPLC	UV and radio	C18 reverse phase	(16,50)
[11C]PBB3	radiochemical and chemical	HPLC	UV and radio	C18 reverse phase	(51)
[11C]UCB-J	radiochemical and chemical	HPLC	UV and radio	C18 reverse phase	(52,53)
N-[11C]meisoindigotin	radiochemical	HPLC	UV and radio	C18 reverse phase	(54)

Table 1 reiterates how standardized C18 HPLC chemical and radiochemical purity QC is for ¹¹C-tracers. A radio detector can refer to either a gamma detector, photon multiplier tube, or NaI crystal, depending on what was used in the reference. There are exceptions where TLC is used. However, USP 823 mandates the quantification of some radiotracers that have mass-dependent localization or toxicity concerns; thus, radio-TLC alone would not suitable for such radiotracers.⁴ The radiochemical purity is simply reported as the percentage of the product peak area on the radio-chromatogram.⁵²

It was found to be a common occurrence to report or estimate the chemical purity of ¹¹C-pharmaceutical preparations containing “unknown materials” or peaks in the HPLC-UV chromatograms, although this is a nonquantitative assessment.²⁹ Additionally, all the radioactivity injected onto the HPLC column might not be recovered, as radiolabeled compounds might not elute or can even irreversibly bind to the packing material of the column.³⁶ Subsequently concerns have been raised that HPLC-UV alone might suitable for the determination of radiochemical purity of all radiotracers.³⁶ Through proper validation procedures, such recovery concerns can be mitigated or identified. Such procedures include the comparison of the injected and eluted radioactivity or by preforming a secondary analysis bypassing the HPLC column and comparing the peak area obtained to asses any possible loss of radioactivity on the column.³⁶

The coelution of contaminants or unknown impurities with the ¹¹C-product is also concerning. Because the HPLC-radiodetector is nonselective and a single channel UV-detector is only partially selective, a second HPLC method (different column and mobile phase) may be considered to verify no coeluting radioactive impurities are present.³⁶

4. Novel or Optimized ¹¹C-Labeled Radiotracer Synthesis

This section compares the development of novel ¹¹C-labeled radiopharmaceuticals in terms of characterization methods and experimental procedures employed to determine if the radiotracer was found to be effective.

Characterization methods refer to analytical methods, such as nuclear magnetic resonance (NMR), that are useful to elucidate the chemical structure of a synthesized compound but are unfeasible to use on every batch of ¹¹C-tracer produced prior to use. Commonly reported characterization techniques include ¹H and ¹³C NMR; mass spectrometry (MS) and high-resolution MS (HRMS). A nonradioactive/cold reference standard is characterized thoroughly and is then used as a reference to the ¹¹C-labeled equivalent compound for the routine QC methodology performed on every batch.

An effective radiotracer should have the following properties: 1) high affinity for the target of interest 2) low background binding or noise 3), and acceptable pharmacokinetic properties to be used in vivo.⁵⁵

There are several physiochemical properties that might be indicative of a promising compound to radiolabel for a potential novel radiotracer such as lipophilicity (expressed as log P_oct/water). log P_oct/water values can be indicative of how the ¹¹C-compound might be absorbed by a biological system or organ from the bloodstream.³⁵ Computational chemistry has also become a popular screening tool to identify not only potential drugs but also radiotracers.⁵⁵

In Table 2, characterization techniques for novel radiotracers are listed along with how radiotracer viability was tested. Additionally, if it was discussed in the publication, the rationale behind the development of the PET tracer was included with the intended target.

Table 2. Novel ¹¹C-Labeled Compounds and Their Employed Characterization Methods.

11C-labeled compound	target and rationale	characterization methods reported in the study	reported assessment of viability	ref
[11C]A1070722	to identify a brain-penetrating GSK3 radioligand	1H NMR	- in vivo primate PET scan	(56)
		HRMS	- 11C-metabolite study with HPLC fractions
		HPLC-UV
[11C]AZ683	AZ683 has a high and selective affinity for Colony Stimulating Factor 1 Receptor (CSF1R).	1H NMR	- in vivo primate and rodent PET scan	(27)
		HRMS
		HPLC-UV
[11C]CMP	11C-structural analogue of a high affinity Glycogen synthase kinase 3 (GSK3) ligand	1H NMR	- in vitro cell uptake assay	(57)
		HRMS	- in vivo rodent microPET
		HPLC-UV
[11C]CPPC	CPPC was demonstrated to have high affinity and specificity for CSF1R.	1H NMR	- in vivo primate and rodent PET scan39	(39,40)
		HRMS	- in vitro cell binding assays
		HPLC-UV	- later clinical study40
[11C]dLop	Identified as a metabolite of [11C]loperamide that showed higher affinity for P-Glycoprotein (P-gp)	1H NMR	- In vivo primate and rodent PET scan	(58,59)
		13C NMR	- 11C-metabolite study with HPLC fractions
		LC-MS/MS	- clinical study58
		HPLC-UV
[11C]6,7-dimethoxy-2-[4-(4-methoxyphenyl)butan-2-yl]-1,2,3,4-tetrahydroisoquinoline	Computer assisted drug design (CADD) docking studies were performed on 46 known to have high σ2 receptor affinity and the most promising compound was selected.	1H NMR	- in vitro cell binding assays	(60)
		13C NMR	- distribution coefficient log D7.4
		LC-MS (ESI)	determined
		HRMS	- ex vivo tissue binding study
			- in vivo rodent PET scan
			- 11C metabolite study with HPLC fractions
[11C]GSK1838705A	GSK1838705A is a high affinity and selective ligand for IGF1R/IR kinase.	1H NMR	- in vivo rodent microPET/CT	(61)
		HRMS	- in vitro study with human cells
		HPLC-UV
[11C]GSK2126458	GSK2126458 is a known PI3K and mTOR kinase inhibitor.	1H NMR	- not assessed in this study	(62)
		HPLC-UV
[11C]HC070	similar chemical structures to existing radiotracers that target TRPC5	HRMS	- in vitro	(26)
		1H NMR	- in vivo rodent and primate PET scan
		13C NMR	- ex vivo biodistribution studies
[11C]HD-800	HD-800 is known to be an inhibitor with high affinity and selectivity of the target colchicine site of microtubules.	1H NMR	- ex vivo biodistribution studies	(63)
		HRMS	- in vivo rodent PET scan
		HPLC-UV
[11C]KSM-01	similar chemical structure to a known selective and high affinity ligand to the PPAR-α target protein complex	1H NMR	- in vivo rodent microPET	(64)
		13C NMR	- in vitro binding study
		HPLC-UV
		melting point
[11C]LAAM	clinically used to prevent opioid withdrawal	HPLC-UV	- in vivo rodent microPET	(65)
			- ex vivo biodistribution
[11C]LY2459989	The authors wanted to develop a more selective radiotracer for κ-opioid receptors.	1H NMR	- in vivo primate PET scan	(66)
		HPLC-MS	- in vitro binding assays
		chiral HPLC-UV	- ex vivo biodistribution rodent studies
[11C]LY2795050	known antagonist κ-opioid receptors	1H NMR	- in vitro binding study44	(43,44,67)
		HPLC-UV	- ex vivo biodistribution44
			- in vivo primate study44
			- later clinical study43,67
[11C]MPC-6827	MPC-6827 has high affinity and selectivity for microtubules.	1H NMR	- lipophilicity determination	(35,68)
		LC-MS/MS	- ex vivo biodistribution
		HPLC-UV	- in vivo rodent microPET/CT
			- in vivo primate study68
[11C]OCM-44	GSK3 inhibitor with high affinity and selectivity. It also has a similar chemical structure to previously reported radiotracers.	1H NMR	- in vivo primate PET69	(69,70)
		13C NMR	- metabolite analysis with HPLC69
			- blocking studies69
		HRMS	- in silico docking study70
		HPLC-UV
[11C]ORM-13070	A radiotracer specific to the α2c adrenoceptor was developed.	HPLC-UV	- in vivo rodent PET/CT	(49,71)
			- ex vivo binding specificity assessment
			- clinical PET71
[11C]ORM-13070- like radiotracers	similar chemical structures to [11C]ORM-13070,identified with CADD docking studies to potentially increase selectivity for the α2c adrenoceptor	1H NMR	-radiochemical stability	(48)
			- in vitro cell binding assay
		13C NMR	- ex vivo biodistribution studies
		LC-MS (ESI)	- in vivo rat studies
N-[11C]meisoindigotin	11C-methylated structural analogue of cancer chemotherapeutic agent	LC-MS/MS	- in vitro lung cancer cell lines	(54)
[11C]PyrATP-1	high affinity and selectivity for the ATP binding site	1H NMR	- in vivo primate and rodent microPET	(72)
		13C NMR	- ex vivo rat brain studies
		HRMS
		HPLC-UV
[11C]RO6931643 and [11C]RO6924963	Both compounds were identified as high-affinity competitors to the target tau aggregate binding site in Alzheimer’s disease.	HPLC-UV	- in vivo primate studies73	(73,74)
		1H NMR	- ex vivo human brain tissue studies73
		LC-HRMS	- passive membrane permeability (PAMPA)74
			- determination of affinity74
			- lipophilicity (log D)74
			- brain lipid membrane binding (LIMBA log Dbrain)74
[11C]UCB-J	UCB-J was identified to be a high affinity ligand to Synaptic Vesicle Glycoprotein 2A in the brain.	1H NMR	- in vivo primate and rodent PET	(53,75,76)
		HRMS	- in vitro cell binding assay
			- lipophilicity (log D7.4) determined
		chiral HPLC-UV	- metabolite analysis with gamma-counter
			- clinical studies75
[11C]VAChT ligand	Twenty new compounds were synthesized and evaluated in vitro, and the compound with the strongest binding affinity for the vesicular acetylcholine transporter (VAChT) was radiolabeled.	X-ray crystallography	- in vitro binding affinities where assayed	(77)
			- lipophilicity (log D7.4) determined
			- in vivo rat and primate studies
			- ex vivo biodistribution
		1H NMR
[11C]WX-132–18B	high-affinity agent for the targeted microtubules	1H NMR	- in vivo rodent microPET/CT	(78)
		HRMS
		HPLC-UV
		melting point

A Google scholar search for ¹¹C-tracer synthesis articles with detailed QC sections, published in the past decade, both in early development, preclinical in vitro, in vivo, and ex vivo cellular and animal studies, and human settings were considered for Table 2.

The term primate, as used in Table 2, can refer to baboons, rhesus monkeys, cynomolgus monkeys, or another species of nonhuman primate used in the referenced study.

Table 2 shows a precedent where usually two or more characterization techniques are reported when a novel radiotracer is synthesized, of which NMR is the most prominent. If the radiolabeled compound is commercially available as a “cold compound”, then a HPLC coinjection or calibration curve is acceptable for characterization of a synthesized radiotracer, although it is seldom that this is reported as the only characterization or qualitative technique employed.

It is apparent that most of the reported characterization (NMR, crystallography, melting point, HRMS) is done on the precursor or preceding intermediates or a cold standard. Given the radiotracer’s half-life, it is logical that only a quick identity confirming test, such as HPLC, can be done on the ¹¹C-tracer. If a reference material is not commercially available, then it seems common practice to synthesize a cold analogue of the radiotracer and to characterize it thoroughly, then later using it as a reference for routine QC. Using a well characterized or certified reference standard is a requirement for clinical productions.⁴ It is theoretically possible to characterize a synthesized ¹¹C-tracers, ¹¹C-byproducts or unknown radio peak after a decay period; for example, if a fraction is collected from the prep-HPLC, it can be analyzed using HRMS in an attempt to identify the compound responsible for the chromatographic peak.

4.1. Rational for choosing a ¹¹C-compound as a potential novel radiotracer

A common trend observed Table 2 is that many novel radiotracers are based on the chemical structures of known inhibitors, ligands, or agents of the target protein or pathway. There are also a number of radiotracers that have a small alteration made to an existing radiotracer in the hope that physiochemical properties will be improved in such a way that the radiotracer exceeds its predecessor in affinity or selectivity for the target receptor. Molecular biology being applied in radiochemistry is common practice and can be seen throughout literature. Knowing the target proteins 3D structure or what compounds can function as inhibitors or ligands with significant affinity is of importance to the development of novel radiotracers.

For a detailed review comparing physiochemical properties of PET tracers, refer to Takamura et al.⁷⁹

The correlation of several physiochemical properties can accurately predict a novel ¹¹C-tracers viability, for instance, the ability of brain lipid membrane binding (LIMBA) to predict nonspecific binding in combination with a high log D_brain coefficient were strongly correlated with the viability of 10 studied radiotracers by Assmus et al.⁸⁰

The data from several in vitro viability tests such as target affinity, density and membrane permeability may be used in conjunction with one another for the selection of the most promising compounds to be further investigated in vivo as potential novel ¹¹C-tracers.⁸⁰

4.1.1. Lipophilicity Values, Biodistribution Coefficients, and LIMBA

There are several variations of lipophilicity values reported in literature; however, the most common reported test was used to estimate how well a radiotracer can penetrate the blood–brain barrier.⁴⁶ The biodistribution coefficient (log D_7.4 value) is a variation on the more widely used 1-octanol/water partition coefficient (log K_OW). For the assessment of a candidate radiotracer, specifically in the field of central nervous system drug development, a liquid–liquid partition coefficient was assessed between 1-octanol and phosphate-buffered saline (pH 7.4). If a large portion of the radiotracer is found in the 1-octanol phase (indicating a high lipophilicity), then the log D_7.4 coefficient should be >1 and is an indication of favorable blood-brain barrier penetration potential for the studied compound.²⁶

Many potential ¹¹C-radiotracers fail due to unfavorably high nonspecific binding to nontarget proteins and phospholipid membranes. LIMBA is useful in assessing the tendency of potential ¹¹C-tracers to undergo nonspecific binding to the brain tissue. The LIMBA test requires a small amount of brain tissue for a label-free assessment of tissue binding. This medium-throughput assay yields a brain tissue/water distribution coefficient.⁸⁰

To demine the concentration of a compound in any of the above-mentioned fractions (water, octanol, or brain tissue) requires the use of HPLC-UV or LC-MS calibration curves.⁸⁰

4.1.2. Computer-aided drug design

Computer-aided drug design (CADD), sometimes referred to as in silico evaluations, is becoming more popular for the identification and screening of potential pharmaceutical compounds. There are two general approaches to CADD referred to as structure and ligand-base screening. The structure-based approach requires knowing the three-dimensional structure of the radiotracers target. An empirical score is calculated as to how well the radiotracer fits to the target with a docking procedure and force field functions. Ligand-based CADD is a pattern recognition technique that focuses on learning relationships between physiochemical properties in candidate radiotracers and a particular experimental value of interest such as binding affinity to the target. Ligand-based approaches work best when there are already a couple of known radiotracers with moderate effectiveness to assesss for similarities.⁵⁵

4.1.3. Binding potential

Binding potential (BP), first conceived in 1984, refers to the affinity of the ¹¹C-compound for its intend target.⁸¹

BP is defined in the above formula, as the ratio of B_max (receptor density) to K_D (¹¹C-compound equilibrium dissociation constant).⁸¹B_max describes the total number of potential receptors in the tissue with which the radiotracer can associate with. The K_D function refers to the ¹¹C-compound’s equilibrium dissociation constant, and is the inverse of the ¹¹C-compound’s affinity for ligand binding, and thus BP. K_D is equal to the ratio of the association and dissociation constants of the ¹¹C-compound to and from the receptors.^79,81

The [¹¹C]raclopride radiotracer was one of the earliest examples of BP assessment and B_max and K_D estimation. This was done through repeated PET imaging, and subsequent plotting of a saturation curve where B_max and the K_D constants could be estimated.⁸²

4.1.4. Displacement Potential

Displacement potential is used to directly compare the affinity of compounds with one another through the displacement of a tritium (³H)-labeled compound from the binding site with the compound to be assessed. For ³H (t_1/2 = ∼12 years) experiments are limited to ex vivo, as the ³H labeled tracer cannot be detected noninvasively.⁷⁹

As a literature example, several potential radiotracers affinity to protein tau aggregates were evaluated in vitro by using human brain sections. The experiment involved the displacement of [³H]T808 from immunohistologically characterized fresh frozen human brain sections derived from AD cases with several candidate compounds for radiolabeling. Two of the tested compounds displaced ∼40% of the [³H]T808 and was found to promising ¹¹C-tracer candidates.⁷⁴

4.1.5. PAMPA

Specifically for ¹¹C-compounds to be used for brain scans, the passive cell membrane permeability assay (PAMPA)⁸³ is indicative of a the potential tracers ability to cross the blood brain barrier (BBB).

PAMPA is used for the evaluation of a compound’s permeability across biological membrane systems in a 96-well sample plate format. In PAMPA, a tracer candidate is allowed to pass through a lipid layer during an incubation period. A sample of each well is then transferred to a UV–vis–compatible microplate and the UV absorption is measured to determine the concentration of the compound that passed through the membrane.^83,84

Recently a real time PAMPA method has been reported in the literature for a high throughput screening of many compounds in a short time. The method uses a fluorescent receptor, composed of a macrocycle combined with a fluorescent dye, administered in the acceptor chamber of a PAMPA microplates. This real time method allows for the differentiation between fast and slow diffusion events.⁸⁴

Further, the results from LIMBA and PAMPA can be used to predict if a ¹¹C-tracer would be affected by the brains efflux transporters.⁷⁴ Correlation between PAMPA and LIMBA indicates a high viability compound for a novel ¹¹C-tracer.⁷⁴

4.2. Viability Assessment of a ¹¹C-Tracer as a Novel Radiopharmaceutical

The gold standard and most employed viability assessment of a new radiotracer is to perform an in vivo preclinical PET scan. This trend is clearly seen in Table 2. It seems to be the absolute exception if an in vivo preclinical PET scan was not reported when a novel ¹¹C-tracer is developed.

There are some other commonly conducted tests, such as in vitro cell binding assays and metabolomics studies using a γ-counter. Especially for rodent studies, an ex vivo bio distribution study is also commonly performed. These additional tests are of importance to understand the biological fate of the radiotracer and can also give insight into the biochemistry that the radiotracer undergoes in vivo.

4.3. Synthesis Optimizations for Established ¹¹C-Tracers

The optimization and atomization of ¹¹C-labeling reactions are an important area of study in the radiopharmaceutical industry to obtain higher molecular activity at injection time or cleaner products.^22,85 Automatization further ensures a radiotracer can be used more broadly on a wider range of synthesis modules.⁶⁵ It is becoming increasingly necessary for PET centers to be more versatile producing several radiotracers and it is impractical to have a dedicated radio synthesizer and hot cell for each individual radiotracer.⁷ Modern commercial radio synthesizers that use disposable cassettes simplify the production of multiple radiotracers on a single unit, without custom tracer specific modifications to the equipment.⁷

Optimizations can have an impact on the QC depending on what was changed in the synthesis process. If new reagents or catalysts are used, they would have to be assessed in the product produced with the new method during QC.

Table 3 indicates that the main reasons for optimizations are to save time and have higher yields and thus specific activity for the radiotracer. Many optimization articles focus on diversifying a radiotracers synthesis procedure to a new module it was not reported on before.

Table 3. Optimizations in Synthesis Procedures for Previously Reported ¹¹C-Tracers.

radiotracer	optimization benefits	confirmation of 11C-tracer identity	ref(s)
[11C]acetate	- higher yield	HPLC-UV with a refractive index detector	(37)
[11C]acetoacetate	- automated on another synthesis unit	HPLC-UV	(85)
	- shorter synthesis time
[11C]choline	- introduction of a different sorbent for SPE	HPLC with a conductivity detector	(22)
	- synthesis on a different module
[11C]UCB-J	- higher yield	HPLC-UV	(38)
	- one step reaction
[11C]PBR28	- higher yields	1H NMR	(86)
	- shorter reaction time	Melting point
		HPLC-UV
[11C]PiB	- higher yields to serve two consecutive patients with a single preparation	HPLC-UV	(87)
	- [11C]CO2 used as synthon	HPLC-UV	(16,88)

Full automation is an exciting development in the PET field, where a tracer production system links multiple modules together, has robotic liquid handlers, grippers, automated fraction collectors, HPLC injection, and automated dispensing allowing for almost exposure-free radiopharmaceutical preparation.³⁸

5. Conclusion

Successful production of a novel ¹¹C-compound to be potentially used as a radiotracer for clinical application requires careful consideration to the implications of QC at each step of the radiosynthesis procedure to prevent contamination or byproduct formation. Selecting a promising compound for ¹¹C-labing, based on its physiochemical properties or known interaction with the target protein complex, is a very important factor for the eventual success in the development of a novel ¹¹C-tracer.

The QC procedures for clinical ¹¹C-tracers are very standardized through mandatory regulation and published guidelines. Additionally, strict precedents have been established for the characterization of novel radiotracers and QC to be employed for animal studies. If a reference material for the proposed novel radiotracer is not commercially available, it is common practice to synthesize a cold analogue of the radiotracer, characterized thoroughly prior to its utilization as a coinjection for rapid HPLC-UV identity confirmation of the radiolabeled compound.

During our literature search, it was made apparent that not all ¹¹C-compound synthesis articles have a detailed section on QC. We reiterate one of Coenen et al. 2017’s statements; all measures used to determine amounts of material (e.g., molar activity or radiochemical purity) should be accompanied by a clear description of the method of its detection. Furthermore, it was not uncommon to come across literature where chemical purity of an ¹¹C-compound was reported using HPLC with UV detection, while the chromatogram contains unknown materials or unlabeled peaks. The determination of a % area using HPLC-UV is a nonquantitative assessment. This issue requires careful consideration and further discussion by the community.

There are many new publications reporting on the development of ¹¹C-compounds for use as radiopharmaceuticals, indicating a thriving research field and community.

Glossary

Abbreviations

¹¹C

carbon-11

BBB

blood–brain barrier

BET

bacterial endotoxin test

CADD

computer aided drug design

CSF1R

colony stimulating factor 1 receptor

cGMP

current good manufacturing practice

CT

computed tomography

EOB

end of bombardment

EOS

end of synthesis

FID

flame ionization detector

GC

gas chromatography

GSK-3

glycogen synthase kinase 3

HPLC

high-performance liquid chromatograph

HRMS

high-resolution mass spectrometry (accurate mass MS)

ICP-AES

inductively coupled plasma atomic emission spectroscopy

LIMBA

brain lipid membrane binding

MA

molar (radio) activity

MS

mass spectrometry (refers to a MS detector on an HPLC)

NMR

nuclear magnetic resonance

OOS

out of specification

PAMPA

passive membrane permeability

PET

positron emission tomography

PI

principle investigator

QC

quality control

RP

responsible pharmacist

SOB

start of bombardment

SOP

standard operating procedure

SPE

solid phase extraction

TLC

thin layer chromatography

UV

ultraviolet (refers to a UV detector on an HPLC)

Author Present Address

^§ MRI Building, G floor, Atrium Health Wake Forest Baptist Medical Center, Winston-Salem, NC 27157, USA

Author Contributions

P.J.M. and K.K.S.S. prepared, reviewed, and edited the manuscript.

The authors are grateful for the funds provided by NIH-NIA: R01AG065839 (K.K.S.S.).

The authors declare no competing financial interest.