With the term “equipment”, it has to be intended all the instrumentation which is involved in the preparation and quality control of radiopharmaceuticals. Their functions, and general principles to be accounted for, will be described in the following two paragraphs, dedicated to the equipment for production and quality control, respectively. Although cyclotrons and nuclear reactors are, strictly speaking, directly involved in the preparation of an essential ingredient, the radionuclide, they will not be covered by the present guidelines, which is also in agreement with Annex 3 – GMP (EU et al. 2017a), that consider this important step in the preparation of RPs as a “non-GMP” step, and as such it’s not requested to be described and justified by the radiopharmaceutical manufacturers. There are practical reasons behind the above choice, that take into account the complexity and multi-tasking intrinsic nature of the radionuclide production equipment/infrastructures. More important, the quality of produced radionuclide(s) is carefully controlled, thus indirectly ensuring that the equipment is working properly and it is producing the intended radionuclide in proper amounts and quality.
Another general comment is related to the software systems, that are integral parts of most of the production and QC equipment, to date. They often play a critical role, performing the following tasks:
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instrumentation control (e.g. running a RP preparation or a dispending sequence in case of automated radiosynthesis or dispensing systems, regulating HPLC pump flowrate or handling the sample injections in case of radio-HPLC, etc.);
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acquisition of data coming from sensors / detectors;
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logging / storage of the acquired data;
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processing of the acquired data and creating reports;
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handle and assure traceability through audit trail functions;
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ensure safety and reliability of the preparations.
For the above reasons, a paragraph will be specifically dedicated to the validation of software and computerised systems, although reference will also be given when necessary throughout the discussion on validation of equipment.
Finally, qualification protocols are aimed to confirm that a system / equipment is properly installed, works correctly and leads to the expected results. This means that the successful outcome of a qualification protocol allows the equipment to be routinely used for the preparation / QC of radiopharmaceuticals, but does not eliminate the need for periodic testing of the instrumentation throughout their life cycle. The type of periodic tests, their recommended frequency and responsibilities are specific for each intended equipment, and they are usually part of the general quality assurance programmes, that should be in place in every radiopharmacy. Often they include tests already performed during the execution of qualification protocols, but that need to be periodically repeated to verify and ensure the correct functionality of the intended equipment. Although their detailed description is out of the scope of the present document, useful reference will be provided in the following paragraphs, especially (but not only) for the routine quality control testing of radioactivity detection and measurement instruments, such as dose calibrators, radio-HPLC “flow” detectors and gamma spectrometers.
Qualification of production equipment
Equipment used in the preparation of RPs usually include: i) radiosynthesis system, which are often, but not necessarily, fully automated; ii) dispensing systems, which are often, but not necessarily, fully automated; iii) suitably shielded hot cells, where radiosynthesis and dispensing systems are located, for radiation protection purposes; telepliers and manipulators are sometime used in those systems not equipped with fully automated devices; iv) hot cells/isolators for manual preparation of RPs (e.g. these are frequently used in the preparation of Tc-99 m labelled kits or in cell labelling); v) dose calibrators. Other instruments or accessories may be used, but they will not be considered in detail by the present guidelines. On the other hand, the same principles and methodologies that will be described for the typical equipment also apply to less frequently used instruments. It has to be considered that production equipment complexity range from relatively simple instruments, such as dose calibrators, to more complicated devices such as automated systems for radiosynthesis or dispensing. Qualification activities should be focused on the most critical components, evaluating the possible effect of failure or miscalibration on the general performance of the system and, in turn, on the quality and safety of the desired RP products.
Installation of production equipment may be preceded by additional evaluation steps, namely Factory Acceptance Testing (FAT) and Site Acceptance Testing (SAT). FAT/SAT are particularly useful in case of complex and/or bulky equipment, or when the intended instrument has been specifically designed, based on URS. A good example is represented by hot cells, whether or not they include automated systems. Hot cells are indeed bulky, and sometimes require some degree of customization, especially in case they have to be used for non-standard processes. During FAT, functionality of major components (buttons, fan, filters, etc.) may be tested, as well as parameters typically verified during OQ such as air velocity, leak tightness or particle contamination. The above tests might help to reveal possible malfunctions or deviations, that may be fixed directly at the Factory, before the shipment. FAT are usually repeated on Site (SAT), and they can be considered as part of the whole qualification.
Radiosynthesis system
“Initial qualification and periodic qualification should be planned in the master document describing each automated module. Initial qualification should include IQ, OQ and PQ. IQ should include the verification of the designed module specifications, the check of installed instrumentation and the integration of working and maintenance instructions in the master document of the module. The functionalities of the automated module without reagents nor chemical components should be checked during OQ, which should also include: i) a verification of the software user access policy, with reference to the different possible level of privileges (e.g. administrators usually have the right to modify any parameters, sequences, methods, etc., while operators should have the possibility to run dispensing programs only); ii) a verification of the software sequences, if applicable; iii) a verification of the possible effects of a general power failure (e.g. to check for the presence and / or the need for an UPS; iv) a verification of the calibration status of the major components; v) a verification of data backup and restore. If the module is a commercial one, the user should ask the supplier to perform a qualification according to internal procedures or to propose a procedure to be performed by the user. If the module is custom made, the user should check that all functionalities, defined in the URS document, meet the specifications included in the master document describing the module. This should include the movement of actuators and the calibration status of the probes (temperature, pressure, and radioactivity). PQ of the module should be conducted by performing three complete runs of a representative process covering all normal operations for the concerned preparation process. For example, a module including a preparative chromatographic system should be qualified selecting a RP preparation process which includes a chromatographic purification. PQ should demonstrate that the module is suitable for the intended application in real conditions of use.
Each automated module should follow a programme of periodic qualifications of the probes (temperature, pressure, and radioactivity) in order to re-calibrate them if needed. For major updates or repairs of the mechanical part, or in case of major modifications of the control software, a risk assessment should be performed in order to evaluate the potential impact on the process performed with the module. OQ and PQ should eventually be performed as conclusions of the risk assessment” (Aerts et al. 2014).
Dispensing systems
General principles outlined for radiosynthesis devices also apply for dispensing systems, which allows for vial or syringe dispensing starting from a “mother” solution prepared using the automated radiosynthesis module. IQ should include: i) a verification of the documentation, such as instruction manuals, drawings, electrical / pneumatic schematics, specifications, certificates; ii) a verification of the major installed components, such as valves, tubing, software, programmable logic controller (PLC) or other suitable control, pumps, balances, control panels, PC, software version, etc. Verification is aimed to check that components are installed in a proper way, by comparison with URS and / or the documentation provided by the manufacturer, if applicable; iii) interconnections between the major components (e.g. cables, tubing assemblies, etc.); iv) a general check of the electrical connections and gas supply piping.
OQ should consider: i) a verification of the software user access policy, with reference to the different possible level of privileges (e.g. administrators usually have the right to modify any parameters, sequences, methods, etc., while operators should have the possibility to run dispensing programs only); ii) a verification of the software sequences, if applicable; iii) a verification of the possible effects of a general power failure (e.g. to check for the presence and / or the need for an UPS; iv) a verification of the calibration status of the major components; for instance, in several dispensing systems, vial filling accuracy is based on balances that weigh the solution during filling operations; balance is in this case a critical component and its performance could be evaluated during OQ by comparison with a calibrated precision balance, using certified weights. Certificate of calibration of the reference balance and weights should not be expired and should be included in the validation documentation. Dispensing systems for individual syringes preparation are preferably based on direct radioactivity determination using dose calibrators: in this case the dose calibrator is the critical component, whose calibration status need to be verified during OQ (see below). One more example of critical components in dispensing systems are the pumps often used to draw / push fluids through tubing assemblies; again, a verification of their calibration (e.g. by measuring dispensed volumes with a reference precision balance) should be performed during OQ; v) a verification of data backup and restore.
PQ of dispensing systems might be carried out by performing at least three successful dispensing cycles in typical working conditions, i.e. using radioactive solutions of the intended activities and radioactive concentrations, dispensed in a representative number of vials / syringes.
Shielded hot cells
Hot cells may be used to accommodate automated or remotely controlled radiosynthesis apparatus or, more simply, to provide the operators a suitable environment to prepare RPs, manually or with the help of tele-pliers, their major functions being to protect the operators from radiation burden (useful calculators to determine the required shielding thickness may be found on the web, see e.g. (Radprocalculator)), and to guarantee an environment with suitable air quality and cleanliness, which is critical for the microbiological quality of the products. Generally, working area is tightly sealed, and a negative pressure is operating, to allow potential radioactive exhaust to be collected to safe containment systems, such as shielded gas cylinders or retardation pipes. Qualification extent for hot cells is dependent on their complexity, that may range from a simple working surface surrounded by an adequate lead shielding, to fully automated dispensing system which are embedded and integrated in the hot cell whole structure. However, there are common characteristics that may allow to set general principles for their validation.
IQ follows the same general concept above depicted for automated systems, and basically consists of a series of verification of the documentation, the major installed components and their interconnections. Specific test for OQ might consider:
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i)
a leak test, to verify the tightness of the working area with respect for the external environment; the test may be performed by simply measuring leak rate after negative pressure has been brought to its maximum, and ventilation / extraction have been switched off, thus isolating the hot cell itself;
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ii)
an air velocity test, to determine the suitability of ventilation above the working area, where RP preparation and dispensing operations take place; an alternative test may be the measurement of air particle contamination, using portable or stand-alone calibrated particle counter devices, which provide and indirect, but nonetheless effective, measure of air quality; indeed, class B or class A environment, as defined by EU GMP – Annex 1 (EU et al. 2017b), are often claimed by hot cell manufacturer. In OQ, these test are performed “at rest”, with working area in normal operating conditions, but without personnel intervention.
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iii)
hot cells doors are usually interlocked for safety reasons; for instance, in case of hot cells used for the preparation of PET RPs, radionuclide transfer from the cyclotron is not allowed if hot cell doors are open; other common safety interlocks link radiation levels inside the working area with hot cell door opening, which is not allowed in case the level is above a defined threshold. Test to verify functionality of interlocks are typical operations to be included in OQ protocols.
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iv)
Other tests, such as laminar flow pattern determination (e.g. using a smoke trace), may be performed, depending on the hot cell characteristics. It has to be underlined once again the need to focus on critical parameters.
It may be appropriate to consider PQ of hot cells in conjunction with OQ, as there is no significant difference in their mode of operation during the preparation of the RPs or at rest. On the other hand, this is not true in case of manual or semi-automated operations, when manipulations may affect laminar flow pattern, e.g. due to the movement of the operating personnel arms through the gloves. Thus, the above test should be executed both at rest (OQ) and “in operation” (PQ). As for particle monitoring, it has to be noted that radioactivity may strongly influence the instrument response, as radiation pulses may be erroneously “counted” by the particle monitoring system, and thus particle contamination may be overestimated. Keeping this in mind, for hot cells dedicated to automated preparation or dispensing operations, PQ tests should be performed during their normal work cycles (e.g. during the preparation or the dispensing of the intended radiopharmaceuticals). In case of hot cells used for manual RP preparations, which are typically performed by personnel through suitable sealed gloves, PQ tests should be performed during normal workflow (e.g. during the preparation of Tc-99 m labelled kits).
Dose calibrators
IQ for dose calibrators should be straightforward, considering that these kind of instruments are relatively simple and consist of a suitable detector connected to a PC or to an electronic control case with a display. Thus, a check of documentation and installation conditions (electrical connections, cabling between detector and measuring / display device) should be sufficient. OQ test should be aimed to verify calibration status of the dose calibrator. This could be done with accuracy and reproducibility tests, to be performed using suitable calibrated radioactivity sources. Chosen radionuclides should be of adequate activity, energy and half-life (e.g. Cs-137,with γ emission at 662 keV, whose T1/2 is = 30 years). OQ might also include other tests, such as verification of calibration through accurate measurements of the output current in response to increasing activities, but usually the monitoring of the above cited parameters is considered sufficient to provide an adequate operational qualification of the instrument. The same test should be performed with the aim of qualifying the performance of the instruments, but using the intended (or one of the intended, in case more radionuclides are measured with the same instrument) radionuclide(s). For instance, in a [18F]FDG preparation facility, accuracy, reproducibility and linearity should be determined using F-18 with activity in the normal working range. For PQ purposes, also Limit of Quantitation (LOQ) should be determined. OQ and PQ tests should take into account the geometry of the sample (e.g. shape and size of the container, and distance to the sensitive surface of the detector). Re-qualification policy of dose calibrators should account that daily checks (e.g. constancy tests) are usually performed, and also verification of linearity and reproducibility are relatively frequent, so as to avoid the need of re-qualification, that should be only done in case the instrument is moved to a different location or due to other significant changes. There are a number of useful reference documents that may help during the implementation of the IQ, OQ and PQ validation steps. Table 6 of EANM guidelines on “Acceptance testing for nuclear medicine instrumentation” (EANM guidelines) provide a list of tests to be performed both at the acceptance of the instrument and to periodically verify its correct functionality. More experimental details related to the above suggested tests are described in EANM guidelines on “Routine quality control recommendations for nuclear medicine instrumentation” (EANM guidelines). Finally, recommendations relevant to assuring the continuing acceptability of the performance of radionuclide calibrators are set by European Commission Radiation Protection document n° 162 “Criteria for Acceptability of Medical Radiological Equipment used in Diagnostic Radiology, Nuclear Medicine and Radiotherapy” (EU Commission & Radiation Protection n. 162).
Qualification of QC instrumentation
Quality control activities may range in complexity from relatively simple test with TLC, which is often the main QC test required in case of Tc-99m labelled kits, to a full series of QC tests, including HPLC, TLC, GC, gamma spectrometry, etc., in case of PET radiopharmaceutical preparations. QC equipment may include instruments normally used in analytical chemistry, such as pH meters, analytical balances, HPLC or GC, and instruments specifically designed for the analysis of radioactive samples, such as activity detectors conjointly used with HPLC or TLC, and gamma spectrometers. Moreover, the need to control microbiological contamination of injectable radiopharmaceutical preparations make devices designed to monitor endotoxin levels familiar to the radiopharmacists.
IQ for QC equipment follows the general rules already depicted for production equipment, and verification of the documentation, drawings, schematics, cables and piping, a general check on SOP, logbook, etc., and verification of environmental conditions and utilities here also apply. OQ and PQ are more specific for the various instruments, and will be described with more details. It has to be underlined once again that IQ, and also OQ, may be also be performed in close cooperation with the instrumentation manufacturer, thus allowing to reduce workload for local radiopharmacy staff.
Radio-HPLC
A radio-HPLC system is typically composed of a pump, which drives the eluent through the various detectors and columns, the detectors themselves, one of which is always a radioactivity detector, while the others are needed to identify and quantify non-radioactive species, and their selection is depending on the intended application. The most frequently used detectors are UV detectors, but conductivity or electrochemical (or others) detectors are also used for specific applications. These detectors will be hereinafter defined as “mass detectors”. Injection of the sample may be performed manually or automatically, by means of an autosampler. Chromatographic columns may be kept at room temperature or heated, by means of a column oven. Finally, most of the HPLC systems currently available are controlled via a suitable software, which is also used to acquire and process signals coming from detectors. From a validation perspective, HPLC may be considered as a sum of different components that may be tested individually. Thus, OQ and PQ test should be designed specifically for e.g. UV detectors, as well as for radiochemical detectors, while control and acquisition software may be evaluated as a whole. OQ on radiochemical detectors may include a linearity verification of the voltage output, in response to decreasing level of radioactivity. A sample of the intended radionuclide/radiopharmaceutical is suitable for this purpose. OQ test on UV detectors usually include: i) test on wavelength accuracy, using a suitable known reference standard; ii) noise and drift test, which can be performed running flow for a suitable time (e.g. 60 min) and recording and allowing software to record the above parameters (some instruments may already have software routines designed to run the tests); iii) a verification of absorbance accuracy using reference standard, which can be easily purchased from commercial supplier, iv) test on software user access and related privileges. Similarly, other “mass detectors” such as conductivity detectors might be OQ checked for linearity and reproducibility using standard ionic solution (e.g. chlorides, sulphates, etc.). HPLC pump may be tested for accuracy and precision by collecting and weighing, using a calibrated analytical balance, a statistically significant number of samples (e.g. 10 samples, collected at a flowrate of 1 ml/min). Column oven, if present, should be checked for its capability to maintain the selected temperature, by setting a range and measuring, using a calibrated thermometer, a range of temperatures. Similarly, accuracy, precision and linearity test might be performed on the autosampler, with the aim to verify their capability to reliably inject samples of the desired volumes. Irrespective of the way the samples are injected (manual or automated), the injection system needs to be cleaned between injections: carry-over is another typical OQ test, aimed to prove the efficacy of the cleaning procedure. Carry-over should be tested by repeatedly analysing samples of mobile phase following the injection of samples containing significant amounts of the intended analytes; to verify carry-over of UV or other “mass detectors”, samples should be taken from the higher concentration solution used in linearity test; for radiation protection purposes, carry-over tests on radiochemicals should be avoided, and the results obtained with test on mass detectors should be considered as sufficient to demonstrate the cleaning efficacy.
PQ protocols on mass detectors should include tests to verify precision (or reproducibility) of the detector output, by injecting at least 5 samples of the intended “cold” counterpart of the desired radiopharmaceuticals (e.g. [19F]FDG for [18F]FDG, or [12C]methionine for [11C]methionine) at a concentration included in the typical working range; also linearity test, using at least 5 different solutions with increasing concentration in the normal working range, should be performed. PQ test on radiochemical detectors should be aimed to check precision and linearity as well. However, due to radioactive decay, a single sample of suitable activity might be used, and area values obtained from the related chromatograms should be recalculated using the decay law (A = A0e-λt). This PQ tests could be considered part of method validation, which will be the subject of a dedicated guideline.
Gas chromatography
In the field of radiopharmacy, gas chromatography is very often (although not always) used for the analysis of residual solvents. The instrument is made of a column (often a capillary column) placed in an oven, through which a carrier gas is swept; the most popular detector is Flame Ionization Detector (FID), which is suitable for residual solvents analysis, but other types may be used, depending on the selected application. Similarly to HPLC, sample injection system may be manual or, more frequently, automated (e.g. head space injection system). IQ protocols don’t differ significantly from those already described for radio-HPLC. OQ tests on FID detectors should include sensitivity. Head space injection system, if present, should be tested for precision and accuracy using reference standard, in order to verify its capability to reliably inject the selected volumes, and for temperature, using a calibrated thermocouple. A leak test, to check the tightness of the injection system, has also to be performed. Finally, test on carry over within the injection system is also recommended. Oven temperature is another critical parameter that should be checked during OQ, by means of a calibrated thermometer; a series of measurements allows for accuracy and precision determination. Also carrier gas flowmeter should be checked, by comparison with a calibrated flowmeter. PQ, as usual, helps to demonstrate that the system is capable to yield the expected performance in normal operating conditions. Precision and linearity should be checked using a reference solution of one or more of the analytes that are expected to be quantified during normal QC operations (e.g. acetonitrile, ethanol), while for linearity determination, a series of solutions with increasing concentrations of the interested analytes should be prepared and analysed. The same data obtained following the above tests, could then be used for the validation of analytical methods.
Radio-TLC
Radio-TLC scanners are mainly used to determine radiochemical purity of radiopharmaceutical preparations. Radio-TLC are often scanners that drive a TLC sheet or plate under a suitable sensor capable to detect radioactivity. Autoradiography systems may also be used for this purpose, that take advantage of the capability of a suitable phosphor plate to store the radioactive signal and release it in the form of a suitable luminescence, and that may thus create a kind of “latent” image of the spots generated during the TLC run by the separation of the analytes. IQ follows the same principles already depicted for other analytical instruments. OQ and PQ may be considered conjointly, and usually tests on reproducibility and linearity, using a solution of the desired radionuclide with suitable activity range should be performed. Reproducibility may be evaluated by deposition, using preferably a calibrated micro-pipette, of a few microliters of the radioactive solution in different position of the TLC plate. During data acquisition and calculations, decay should be accounted for, especially in case of very short half-life radionuclides. For linearity purposes, a single spot could be deposited and acquired at suitable user defined intervals. Other OQ tests may be related, as usual, to the software system, by checking software access policy and privileges, and archiving/backup functions.
Gamma spectrometer
Gamma spectrometers are used both for identification and radionuclidic purity determination purposes. Most common detectors are HPGe (high purity germanium), which are to be preferred due to higher energy resolution, and thallium activated NaI detectors, that have better sensitivity but much lower energy resolution. Detectors are suitably shielded, to reduce background, and connected through a cable to a PC equipped with a proper software. HPGe also need to be cooled, in order to reduce thermally induced leakage current, and effectiveness of the cooling system, irrespective if this is done via liquid nitrogen or electrically, has to be carefully controlled during normal operations and during qualification tests as well. With such a simple configuration, IQ is also quite simple, and it may be performed, as already described for other instruments, by a general check of the documentation (order, manuals, other documents provided by the supplier, logbook, dedicated SOPs, installed software, versions, etc.). OQ may include an energy calibration of the instrument, with the aim to verify that detected energies match with expected values. Both mono- and multinuclide calibration sources may be used. Multinuclide or single-nuclide multi-energy (e.g. Eu-152) sources should be preferred, so as to check calibration status in a broader energy range. Usually, typical working range of the above instruments is indeed 0–2000 KeV. A minimum of 6 acquisitions for each of the selected energy signals, followed by coefficient of variation (CV%) calculation allow for energy calibration determination. Efficiency is another parameter to be considered in OQ, especially when gamma spectrometry is used for quantification purposes. Here also multinuclide sources are ideally suited, as they allow for quantification of radioactivity amount of the various nuclides, provided that they are sufficiently long lived (medium half-life radionuclides might also be used, but errors are higher). PQ is depending on the intended use of the instrument, but it generally includes reproducibility and linearity tests, to be performed with the radionuclides expected in the RP preparation of concern. The sensitivity of an instrument is usually measured, as already described above, using calibrated standards at the proper concentration. In case of gamma spectrometer, sensitivity may be expressed by a parameter known as Minimum Detectable Activity (MDA), which may be considered similar to the Limit of Detection (LOD), and which is dependent on many factors (background, geometry, etc.) and it may vary from run to run for the same radionuclide. Thus, although MDA might be determined, for example, during OQ test with calibrated source(s) or during PQ with the intended radionuclide, it would make more sense to evaluate it during validation of the specific analytical method. It is also important to establish the maximum detectable activity range, as the saturation of the detector may lead to underestimation of the radioactivity.