Influence of the Generator in-Growth Time on the Final Radiochemical Purity and Stability of ^99mTc Radiopharmaceuticals

Abstract

At Legnaro laboratories of the Italian National Institute for Nuclear Physics (INFN), a feasibility study has started since 2011 related to accelerated-based direct production of ^99mTc by the ¹⁰⁰Mo(p,2n)^99mTc reaction. Both theoretical investigations and some recent preliminary irradiation tests on ¹⁰⁰Mo-enriched samples have pointed out that both the ^99gTc/^99mTc ratio and the ^99mTc specific activity will be basically different in the final accelerator-produced Tc with respect to generator-produced one, which might affect the radiopharmaceutical procedures. The aim of this work was to evaluate the possible impact of different ^99gTc/^99mTc isomeric ratios on the preparation of different Tc-labeled pharmaceutical kits. A set of measurements with ^99mTc, eluted from a standard ⁹⁹Mo/^99mTc generator, was performed, and results on both radiochemical purity and stability studies (following the standard quality control procedures) are reported for a set of widely used pharmaceuticals (i.e., ^99mTc-Sestamibi, ^99mTc-ECD, ^99mTc-MAG3, ^99mTc-DTPA, ^99mTc-MDP, ^99mTc-HMDP, ^99mTc-nanocolloids, and ^99mTc-DMSA). These pharmaceuticals have been all reconstituted with either the first [^99mTcO₄]⁻ eluate obtained from a ⁹⁹Mo/^99mTc generator (coming from two different companies) or eluates after 24, 36, 48, and 72 hours from last elution. Results show that the radiochemical purity and stability of these radiopharmaceuticals were not affected up to the value of 11.84 for the ^99gTc/^99mTc ratio.

1. Introduction

^99mTc, with its peculiar physical-chemical properties, still continues to be the most important radionuclide used in diagnostic nuclear medical procedures. In particular, the developments of technetium chemistry have opened new perspectives in the field of diagnostic imaging [1]. More than 80% of the radiopharmaceuticals are currently labeled with this radionuclide [1] by reconstitution with sodium pertechnetate [2–4] [Na^99mTcO₄] commercial kits containing in lyophilized form the various reagents required for the preparation of each radiopharmaceutical. Its routine applications are ensured by the availability of portable ⁹⁹Mo/^99mTc generators in which ⁹⁹Mo is bound as molybdate anion to alumina columns. Current global interruptions of ⁹⁹Mo supply that involved uranium fission of highly enriched ²³⁵U targets, aging reactors, and the staggering costs of their maintenance, focused on the search for alternative method of the ^99mTc production [5]. One of the possibilities is to replace the reactors with particle accelerators, aiming at a regional production and distribution. At Legnaro laboratories of the Italian National Institute for Nuclear Physics (INFN), a feasibility study related to accelerated-based direct production of  ^99mTc by the ¹⁰⁰Mo(p,2n) ^99mTc reaction [6, 7] has started since 2011. Theoretical investigations and some recent preliminary irradiation tests on ¹⁰⁰Mo-enriched samples point out that both the ^99gTc/^99mTc ratio and ^99mTc specific activity will be basically different in the final accelerator-produced Tc with respect to generator-produced one, due to the concomitant production of Tc contaminant nuclides, such as ^99gTc, ⁹⁸Tc, ^97mTc, and ^97gTc. In particular, the amount of the ground-state long-lived β⁻  emitter ^99gTc, useless for diagnostic procedures, might have a negative effect in the radiopharmaceutical procedures going to compete with ^99mTc for the formation of the corresponding chemically identical radiopharmaceuticals. The presence of an excess of ^99gTc might be responsible for a value of radiochemical purity lower than the standard required for some radiopharmaceutical preparations. In fact, the ^99gTc present in solution could consume reagents of reaction, and in particular the reducing agent (SnCl₂). As a result, unreacted [^99mTcO₄]⁻ may remain in the solution, or radioactive by-products not useful for the specific diagnostic procedure may be formed. The quality of ^99mTc is then fundamental for the assurance of radiopharmaceuticals quality [8–10]. The aim of this work was therefore to perform a set of measurements with ^99mTc, eluted from a standard ⁹⁹Mo/^99mTc generator, in order to first check possible impact of different ^99gTc/^99mTc isomeric ratios on the preparation of different Tc-labeled pharmaceutical kits. Results on both radiochemical purity and stability studies (following the standard quality control procedures) are reported for a set of widely used pharmaceuticals (i.e., ^99mTc-Sestamibi, ^99mTc-ECD, ^99mTc-MAG3, ^99mTc-DTPA, ^99mTc-MDP, ^99mTc-HMDP, ^99mTc-nanocolloids, and ^99mTc-DMSA). These pharmaceuticals have been all reconstituted with either the first [^99mTcO₄]⁻ eluate obtained from the ⁹⁹Mo/^99mTc generator (coming from two different companies) or eluates after 24, 36, 48, and 72 hours from last elution.

2. Materials and Methods

The preparation of radiopharmaceuticals reported in Table 1 was carried out with sodium pertechnetate eluates coming from two different ⁹⁹Mo/^99mTc generators: a “dry” DRYTEC generator (GE Healthcare, Via Galeno 36 20126, Milan), and a “wet” Elumatic III generator (IBA-CIS Bio International, Route Nationale 306, Saclay BP 32, 91192 GIF SUR YVETTE, Cedex France).

Table 1 

Radiopharmaceuticals used in the study.

All generators, with ⁹⁹Mo calibrated activity of 10 GBq, were eluted with 5 mL of saline solution as indicated by each manufacturer. From each generator, we analyzed and compared the three first elutions, performed just after generator delivery (time elapsed between manufacturing and first use can be estimate in 2-3 days), and 3 elutions were carried out after 36, 48, and 72 hours from the previous elution.

2.1. Quality Control of  ⁹⁹Mo/^99mTc Generator Eluates

Generator eluates have been subjected to all the tests [11] required by European Pharmacopoeia [14], and Italian Pharmacopoeia, 12th edn., Norme di Buona Preparazione dei Radiofarmaci per Medicina Nucleare, All. A, p.to A.2 “Generatore di ⁹⁹Mo/^99mTc (molibdeno/tecnezio)”.

2.1.1. Generator Elution Yield

It was expressed as % of the ratio between the eluate radioactivity measured immediately after elution using a dose calibrator (𝑉_𝑀) and the theoretical radioactivity (𝑉_𝐶) calculated on the basis of the date of calibration and multiplied by the factor 100 [3]. The elution efficiency should be within the range of 90%–110%.

2.1.2. Eluate Visual Inspection

All eluates were visually inspected, pulling the vial from its shielded container. The operation was performed within an adequately shielded cell for radiopharmaceuticals manipulation; the vial containing the pertechnetate eluate was manipulated by operators using a pair of pliers to guarantee an adequate distance from the hands of the operator.

2.1.3. Eluate pH

Generator eluate is itself a preparation for injection; ideally it should have a pH as close as possible to the physiological, between 7 and 8. The Pharmacopeia requires that eluates have pH values within the range of 4–8. Since the molybdenum is adsorbed onto the alumina in an acid environment, the pH values of eluates are slightly acid (4.5–6). It was measured by means of pH usual indicator strip (range 0–14) and checked by electronic pH-meter.

2.1.4. Aluminum Content

It was determined by a semiquantitative procedure employing indicator strips (Tec-Control Biodex Medical, New York, USA) together with standard aluminium solution [12, 13]. A drop of standard solution with a concentration less than 5 μg mL⁻¹ of aurintricarboxylic acid was deposited on indicator paper; subsequently, by side a drop of eluate was deposited. If the coloration produced by the latter is lower than the one produced by the standard solution, it can be assumed that the concentration of aluminium in the eluate is less than the maximum acceptable level of 5 μg mL⁻¹ provided by Official Pharmacopoeia [14].

2.1.5. Radionuclidic Purity

Radionuclidic purity is the percentage of total radioactivity which may be attributed to the daughter radionuclide. In the case of fission-produced generator, the largest potential source of contamination that could exceed the minimum value detectable can be due to the parent (⁹⁹Mo) [15]. Trace amount of other fission impurities [16, 17] may be usually present in negligible amounts.

The early and quick evaluation of the eluate content of  ⁹⁹Mo was provided by the following procedure, which involved the use of a lead shield of appropriate thickness (0.6 mm of lead) in order to attenuate ^99mTc emission [18]. The activity contained in the unshielded elution vial was measured with a dose calibrator (PET-dose, Comecer, Castelbolognese, Italy); for measuring ⁹⁹Mo activity, the elution vial was then placed within the lead shield and its activity was recorded. The thickness of the shield was enough to largely attenuate the 140 keV photons [19], and only partly those greater than 700 keV. The measured activity, multiplied by a suitable correction factor which accounts for the attenuation of 740–780 keV photons due to the lead shielding, provides an estimation of the ⁹⁹Mo activity. This value should not exceed 0.1% of the ^99mTc activity according to the European Pharmacopoeia.

For a more accurate determination of the radionuclidic purity [17, 20], the same eluate sample was examined again (reassayed) after 7–15 days by means of high-resolution gamma spectrometry using a solid-state, high-purity germanium detector [16].

2.1.6. Radiochemical Purity

It was checked by paper chromatography, Whatman no.1 paper strips and saline as mobile phase. According to this procedure, ^99mTcO₄⁻ migrates with the solvent front (𝑅_𝑓=1), whereas reduced hydrolyzed ^99mTc remains at the origin (𝑅_𝑓=0). The radioactivity distribution was measured by a scanning radiochromatography detection system for thin layer chromatography (Cyclone instrument equipped with a phosphor imaging screen and an OptiQuant image analysis software (Packard, Meridien, CT)). Eluate radiochemical purity should be greater than 95%.

2.1.7. ^99gTc to Active ^99mTc Ratio

⁹⁹Mo decays to ^99gTc (12.4%) and ^99mTc (87.6%), and the latter, with a physical 𝑇_1/2 of 6.0067 h, decays to ^99gTc (𝑇_1/2 = 211,100 years). Due to this particular branching decay of  ⁹⁹Mo, even fresh elutions from a generator always contain both isotopes (^99mTc and ^99gTc), indistinguishable from the chemical point of view. The amount (μg) of total technetium present in the eluate is directly related to the amount of ⁹⁹Mo atoms present on the column (i.e., ⁹⁹Mo activity) and the time that elapsed since the previous elution. The total number of Tc atoms, namely, the sum of  ^99gTc and ^99mTc, has been calculated as follows: 𝑁_Tc(total)=𝑁^∘_99Mo1−𝑒^−𝜆1𝑡, (1) where 𝑁^∘_99Mo is the initial ⁹⁹Mo atoms number present on the column, 𝜆₁ is the decay constant of  ⁹⁹Mo (0.0105 hours⁻¹), and 𝑡 is the time that elapsed since the last elution. The number of ^99mTc atoms has been calculate as follows: 𝑁_99mTc𝜆=𝑃¹𝜆₂−𝜆₁𝑁^∘_99Mo𝑒^−𝜆1𝑡−𝑒^−𝜆2𝑡, (2) where 𝜆₂ is the decay constant of  ^99mTc (0.1149 hours⁻¹) and 𝑃 is the decay probability (𝑃=0.876). A simplified decay scheme of  ⁹⁹Mo to ^99gTc is shown in Figure 1. From the above equations the number of  ^99gTc atoms can be easily calculated as follows: 𝑁_99gTc=𝑁_TotalTc−𝑁_99mTc (3) and thus the ^99gTc to active ^99mTc ratio can be estimated.

Figure 1 

Simplified decay scheme of 99Mo to 99gTc.

The determination of the ^99gTc content in a fresh eluate requires an immediate measurement after the elution of the ^99mTc activity and a later measurement of the total activity of ^99gTc (in a few months almost all the ⁹⁹Mo and ^99mTc atoms decay into ^99gTc). The evaluation of ^99mTc activity in the sample has been performed by using a dose calibrator (PET-dose, Comecer, Castelbolognese, Italy), while the evaluation of ^99gTc activity has been performed using the TRI-CARB 2810TR liquid scintillation analyzer (Perkin Elmer Inc., Monza, Italy). The samples for ^99gTc activity measurements were prepared taking an aliquot of 0.8 mL from an eluate decayed for 60 days (total volume of the eluate: 5 mL) and adding 5.4 mL of liquid scintillator (Ultima Gold LLT cocktail, Perkin Elmer Inc., Monza, Italy). The measurement of ^99gTc activity was performed using the 0–295 keV energy window.

2.2. Radiopharmaceuticals Labeling

The elutions were used to label different commercial kits (Table 1). Kits reconstitution was performed according to the methods described in the package included within the commercial kits. The radiochemical purity (RCP) of radiopharmaceuticals was evaluated immediately after preparation (𝑡=0) and at the end of the stability period indicated by the manufacturer. The radiochemical purity and stability were measured using methods specified by manufacturer, with the exception of TechneScan (Mallinckrodt) for which the following chromatographic system was used [21]: mobile phase, 54/45/1 (physiological/methanol/glacial acetic acid) and stationary phase, RP-18 (Merck). Thin-layer chromatography plates were analyzed with a Cyclone instrument equipped with a phosphor imaging screen and an OptiQuant image analysis software (Packard, Meridien, CT).

2.3. Imaging Studies

Three ^99mTc eluates produced by ⁹⁹Mo/^99mTc generator with different ^99gTc/^99mTc ratio 𝑅 were used for imaging studies; the 𝑅 values were 4.16, 9.51, and 15.2, respectively. Each tomographic acquisition has been performed by filling a NEMA phantom NU 4-2008 with 74 MBq of ^99mTc-pertechnetate solution, and the data have been acquired with the YAP-(S)PET small animal scanner prototype [22] and reconstructed by using an EM-ML algorithm.

3. Results and Discussion

The results of the quality control (Table 2) performed on all eluates obtained from two different generator (DRYTEC GE Healthcare and Elumatic III IBA) are consistent with the European Pharmacopoeia requirements [12]. For simplicity, data of visual inspection, yield of elution, and the aluminum content in eluates are not reported, because they fell within European Pharmacopoeia requirements. The radiochemical purity (RCP) values of all radiopharmaceuticals labeled with each eluate are reported in Tables 3 and 4. Results refer to the RCP evaluated immediately after the preparation (𝑡=0). For simplicity, data at the end of the stability period specified by the manufacturer are not reported, because they fell within the specifications required by the manufacturer. Tables 5 and 6 report the RCP data obtained from reconstitution of the kits with the first eluate. The values ​​refer to the checks carried out immediately after the preparation (𝑡=0) and at the end of the stability period specified by the manufacturer in the package insert of each radiopharmaceutical. The values ​​of radiochemical purity are always superior to the standards required by the manufacturer. The results show that the total amount of technetium (^99gTc + ^99mTc) present in the first eluate and in the eluates obtained at longer intervals, from 24 h up to 72 h, did not affect the radiochemical purity of the final products. Table 7 shows an estimation of the total amount of technetium present in an eluate obtained from a ^99mTc generator with ⁹⁹Mo calibrated activity of 10 GBq. The ratios 𝑅 of three ^99mTc eluates at 24 hours and two ^99mTc first eluates at 48 hours have been measured, and the results have been 𝑅_24h=3.23±0.15 and 𝑅_48h=6.68±0.31, respectively. While the experimental value of first eluates at 48 hours is in good agreement with the theoretical value of 6.5, the experimental value of eluates at 24 hours shows a large difference with respect to the theoretical value of 2.55. This discrepancy could be explained by taking into account the elution efficiency 𝜀=0.91 of ⁹⁹Mo generators used in our work. Indeed, the recalculated ratio 𝑅 at 24 hours is included in the range (2.78–3.38) and depends on temporal sequence of previous elutions. The reconstructed SPECT images of NEMA phantom, for the different ^99gTc/^99mTc ratios 𝑅 used, are shown in Figure 2. The average reconstructed activity along the phantom axis, for the three values of 𝑅, is shown in Figure 3. The visual inspection on the images doesn’t show significant difference in image quality and radioactivity distribution. Currently, CERETEC is the only commercial product for which the use of a fresh eluate, obtained from a generator eluted for not more than 24 hours, is required. This exception is linked to the low amount of tin chloride dehydrate in its formulation (7.6 μg), which makes its radiochemical purity strongly influenced by the amount of ^99gTc present in the eluate. All formulations studied possess significantly higher amount of tin. A further limitation to the use of eluates characterized by greater amount of ^99gTc (elution intervals > 24 h) could be due to radioprotection reasons related to the physical characteristics of ^99gTc (𝑡_1/2=2×10⁵ y, 𝛽_max = 292 keV). However the amount of radioactivity associated with the mass value is very low (e.g., 1 μg of  ^99gTc corresponds to 630 Bq).

Table 2 

pH, ⁹⁹Mo, ¹⁰³Ru, and ¹³¹I amounts and radiochemical purities (mean ± standard deviation) of the evaluated generators’ elutions.

Table 3 

RCP of radiopharmaceuticals at 𝑡=0, prepared with generator DRYTEC GE Healthcare eluates at time superior to 24 h from the last elution.

Table 4 

RCP of radiopharmaceuticals at 𝑡=0, prepared with generator Elumatic III (IBA) eluates at time superior to 24 h from the last elution.

Table 5 

RCP of radiopharmaceuticals at 𝑡=0 and at the expired time specified by the manufacturer, prepared with the first eluate obtained from DRYTEC GE Healthcare generator.

Table 6 

RCP of radiopharmaceuticals at 𝑡=0 and at the expired time specified by the manufacturer, prepared with the first eluate obtained from Elumatic III (IBA) generator.

Table 7 

Evaluation of total technetium amount in ^99mTc eluates coming from a generator with ⁹⁹Mo calibrated activity of 10 GBq, at different times by previous elution.

(a)

(b)

(c)

Figure 2 

Reconstructed SPECT trans-axial slices of NEMA NU 4-2008 filled with 99mTc-pertechnetate solution. Top images (a) have 𝑅=4.16, middle images (b) have 𝑅=9.51, and bottom images (c) have 𝑅=15.2.

Figure 3 

The average activity for reconstructed 40 slices of NEMA NU 4-2008 phantom filled with 99mTc-pertechnetate solution having 𝑅=4.16,  𝑅=9.51, and 𝑅=15.2.

4. Conclusion

In order to first check the possible impact of different ^99gTc/^99mTc isomeric ratios on the preparation of different Tc-labeled pharmaceutical kits, the radiochemical purity and stability of ^99mTc-Sestamibi, ^99mTc-ECD, ^99mTc-MAG3, ^99mTc-DTPA, ^99mTc-MDP, ^99mTc-HMDP, ^99mTc-nanocolloids, and ^99mTc-DMSA were studied by using ^99mTc eluates coming from ⁹⁹Mo/^99mTc generator eluted at different times from the previous elution. The results prove that radiochemical purity and stability of these radiopharmaceuticals are not affected up to ^99gTc/^99mTc ratio of 11.84.

A future goal will be to repeat the experiments with ^99mTc eluates coming from generators with ⁹⁹Mo calibrated activity higher than 10 GBq, in order to check the possible impact of  ^99gTc in higher ^99mTc activities solutions at different ^99gTc/^99mTc ratio.

Another future goal will be to study the impact of accelerated-based ^99gTc and other Tc-isotopes on the image quality and determine the allowed limit for ^99gTc and other Tc-isotopes in the final accelerator-produced Tc.

Conflict of Interests

The authors declare that they have no conflict of interests.

Acknowledgments

The authors would like to thank to Dr. L. M. Feggi (Unit of Nuclear Medicine, Sant’ Anna Hospital, Ferrara) for equipment and IBA-CIS Bio International, GIF SUR YVETTE, Cedex France for generators.