- Email: [email protected]
Use of 65Zn as a tracer for the assessment of purification in the 68Ga-DOTANOC synthesis
Applied Radiation and Isotopes 80 (2013) 27–31
Contents lists available at SciVerse ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Use of 65Zn as a tracer for the assessment of purification in the 68Ga-DOTANOC synthesis G. Lucconi a,b, G. Cicoria b, D. Pancaldi b, F. Lodi c, C. Malizia c, S. Fanti c, S. Boschi c, M. Marengo b,n a
Postgraduate School in Medical Physics, University of Bologna, Italy Medical Physics Department, S.Orsola-Malpighi University Hospital, Bologna, Italy c Nuclear Medicine Unit, S.Orsola-Malpighi University Hospital, Bologna, Italy b
H I G H L I G H T S
We studied 68Ga-DOTANOC radionuclidic purity and 68Zn distribution in the synthesis. 68Zn behavior was inspected by using cyclotron produced 65Zn as a radiotracer. 68Zn competes with 68Ga in labeling DOTANOC with a (95 7 2)% labeling yield. Effectiveness of the STRATA X-C cationic cartridge in lowering the amount of 68Zn to less than 1%. 68Ga-DOTANOC radionuclidic purity was (99.9999986 70.0000006)%, superior to required 99.9%.
art ic l e i nf o
a b s t r a c t
Article history: Received 31 August 2012 Received in revised form 17 April 2013 Accepted 2 May 2013 Available online 30 May 2013
In the last years 68Ga has got into the focus of researchers and clinicians especially for radio-labeling of biomolecules; an important characteristic of this positron emitting isotope is its availability via the 68 Ge/68Ga generator system: the long-lived 68Ge (t1/2 ¼270.8 d) produces the short-lived 68Ga (t1/2 ¼ 67.63 min) which decays to stable 68Zn. 68Ge breakthrough compromises 68Ga radionuclidic purity, while 68 Zn might affect the specific activity of the radiopharmaceutical. In this paper we investigated the weight of these impurities in 68Ga-DOTANOC synthesis. 65 Zn (t1/2 ¼ 244.26 d; decay mode: EC 98.3%, β+ 1.7%) was used as a radiotracer of stable 68Zn; samples of the purification columns, wastes and product were recovered and measured with a calibrated HPGe gamma-ray spectrometry system. The results showed that 68Zn competes with 68Ga in labeling DOTANOC with a (957 2)% labeling yield; they also proved the effectiveness of the STRATA X-C cationic post-processing of the generator eluate in lowering the amount of this impurity to less than 1%. Moreover this approach, along with the purification of the final product through a STRATA X cartridge, effectively removes 68Ge breakthrough providing a 68Ga-DOTANOC radionuclidic purity of (99.9999986 7 0.0000006)%, superior to 99.9% required by the Pharmacopoeia Monograph on 68Ga Edotreotide injection. & 2013 Elsevier Ltd. All rights reserved.
Keywords: 68 Ga-DOTANOC 65 Zn Radioactive tracer Specific activity Radionuclidic purity
1. Introduction Positron-emission tomography (PET) is a well-established diagnostic imaging technique in nuclear medicine; it combines high sensitivity and relatively high resolution, therefore allowing early biochemical assessment of pathology. The recent development of hybrid PET/CT scans has allowed an integration of functional imaging and anatomical localization at a superior
n
Corresponding author. Tel.: +39 051 6363575; fax: +39 051 6363571. E-mail address: [email protected] (M. Marengo).
0969-8043/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.apradiso.2013.05.006
spatial resolution, consequently changing many fields of medical diagnosis. The most frequently used positron emitters are 18F, 11C, 13N and 15 O, produced in a cyclotron through the irradiation of liquid and gaseous targets (Finn and Schlyer, 2001). 68Ga is a positron emitter of great interest because of its physical half-life which is compatible with the pharmacokinetics of most radiopharmaceuticals of low molecular weight; besides it is available via 68Ge/68Ga generators which avoid the need for an on-site cyclotron (Fani et al., 2008). This radionuclide (t1/2 ¼67.63 min; decay mode: 89% β+, 11% EC; Emax(β+)¼1.90 MeV (Lund Nuclear Data Service, 2007)) is indeed conveniently produced exploiting the secular equilibrium
28
G. Lucconi et al. / Applied Radiation and Isotopes 80 (2013) 27–31
with the parent radionuclide 68Ge (t1/2 ¼ 270.8 d; decay mode: 100% EC (Lund Nuclear Data Service, 2007)), trapped on an inorganic or organic resin (Roesch and Riss, 2010). The long halflife of the latter grants 68Ga availability for long periods of time and allows to elute the generator twice a day. Recently, 68Ga-DOTA labeled peptides have become the gold standard for the evaluation of neuroendocrine tumors (NET) with PET/CT, being superior to conventional imaging modalities and the previous 111In-Octreotide SPECT technique (Ambrosini et al., 2010). Despite the convenient availability, most of the 68Ge/68Ga generator systems are yet not optimally designed for direct radio-labeling because of the low pH and large volume of the eluate (Asti et al., 2008); in addition the solution contains measurable activities of long-lived 68Ge and other metal impurities such as 68Zn produced by 68Ga decay. 68Ge affects the radionuclidic purity of the radiopharmaceutical; stable 68Zn, by competing with 68Ga in the labeling reaction, may compromise its specific activity which should be kept as high as possible in order to maximize the tumour-to-background ratio. The DOTA conjugated peptide DOTANOC binds to 68Ga with a ligand to metal ratio 1:1; since 1 nmol of 68Ga holds an activity of 98 GBq, the theoretical specific activity of 68Ga-DOTANOC is 98 GBq/nmol (Zhernosekov et al., 2007). Yet this value may be lowered by the presence of contaminants competing with 68Ga for incorporation in DOTA, such as Zn2+ and Fe2+/3+ (Breeman et al., 2005). In this study we investigated the efficiency of the cationic postprocessing of the 68Ga-eluate implemented in the Eckert & Ziegler Eurotope module for 68Ga DOTA conjugated peptides synthesis. Particular attention was directed to the investigation of 68Zn relative distribution in the different steps of the synthesis using 65 Zn, produced at our laboratory, as a radiotracer. This choice allowed the study of 68Zn distribution through radiometric measures available in most PET centers, thus avoiding more complex and expensive analysis with mass spectrometer devices.
2. Materials and methods 2.1.
68
Ga production
68 Ga was obtained from two in series 68Ge/68Ga generators (Eckert & Ziegler Eurotope, distributed in Italy by Radius, Budrio, BO), each with an initial activity of 1850 MBq at the calibration date, with a difference of nine months between the two generators. The certified initial elution yield was (76.3 70.9)% and (86 74)% and was supposed to remain greater than 70% for the following nine months; 68Ge breakthrough resulted less than 5 10−3% of the total eluted activity.
2.2.
68
68
Ga-DOTANOC synthesis
Ga-DOTANOC syntheses were performed with the (Eckert & Ziegler) Modular Lab automated system. DOTANOC precursor (ABX GmbH, Radeberg, Germany) and all the other reagents were of pharmaceutical grade. Two in series 68Ge/68Ga generators were eluted with 15 ml of 0.1 N HCl at a flow of 2 ml/min using a remote controlled pump (Fig. 1). The eluate was loaded onto a disposable Phenomenex STRATA X-C cationic exchange cartridge in order to remove 68Ge breakthrough and other metal impurities in the “generator waste”. In a second step the column was washed with 0.8 ml of 98% acetone/2% 0.02 N HCl solution to recover trapped 68Ga, which was directly transferred to the reactor vessel, containing 40 mg of DOTANOC diluted in a 0.2 N sodium acetate/acetic acid buffer, pH
(4.070.2). The solution was heated to 90 1C for 5 min and then loaded onto a pre-conditioned Phenomenex STRATA X reversed phase cartridge for purification from unreacted 68Ga, collected in the “synthesis waste”. 68Ga-DOTANOC was finally recovered with 0.8 ml of ethanol, diluted with 7.0 ml of saline and then sterilized with a 0.22 μm filter (Ocak et al., 2010). The activity of the labeled solution was measured at the End of Synthesis (EOS) with a CRC15 PET ionization chamber radionuclide activity meter (Capintec Inc., Ramsey, NJ) calibrated by means of NIST traceable standard (mod. BM06S-685, RadQual Inc., Weare NH).
2.3.
5
Zn production and application
Despite the large availability of 65Zn solutions, we used 65Zn previously produced at our laboratory via the 65Cu(p,n)65Zn nuclear reaction using a 16.5 MeV GE-PET trace biomedical cyclotron (Lucconi et al., 2012). Natural copper foils (thickness: 100 μm; purity: 499.9%) were irradiated in a solid target station developed in our institution; 65Zn was purified from copper and other long-lived contaminants through a BIO RAD AG1-X8 anionic exchange resin 100–200 mesh Cl− form (Bio-Rad Lab., California, USA) and finally transferred to a 0.1 N HCl solution suitable for radiopharmaceutical synthesis. The radionuclidic purity of the tracer, expressed in activity, was 4(99.921 70.003)%. 65 Zn solution was used as a tracer of 68Zn in different steps of the synthesis: − The elution yield of 68Zn from a 68Ge/68Ga generator: a single Eckert & Ziegler 68Ge/68Ga decommissioned generator was eluted with 1 ml of 65Zn solution holding an activity of (25.3 71.0) kBq, followed by 9 ml of 0.1 N HCl in order to simulate standard elution volume (10 ml of 0.1 N HCl). The generator was then regularly eluted twice as to study the eventual retention of 65Zn on the resin. The eluates were investigated through gamma-ray spectrometry. − The elution profile of 68Zn from a 68Ge/68Ga generator: the death volume of the same decommissioned generator was measured with 0.1 N HCl; 1 ml of 65Zn solution was then loaded followed by enough air to place the tracer in the middle of the death volume, where the resin was supposed to be. The generator was eluted with 15 ml of 0.1 N HCl and the elution profile was sampled with 1 cm3 tubes. 68Ga activity was measured with a CRC15 PET ionization chamber radionuclide activity meter (Capintec Inc., Ramsey, NJ), while 65Zn activity was measured with gamma ray spectrometry after substantial 68Ga decay in order to improve the accuracy of the measurement. − Purification with the STRATA X-C cationic exchange cartridge: 5 tests were performed in order to investigate the low affinity of the resin with zinc. 1 ml of 65Zn solution was loaded onto a STRATA X-C column in standard synthesis conditions. The column was then eluted with 9 ml of 0.1 N HCl and taken to dryness with a 30 s nitrogen flow, while collecting the solution in the “generator waste”. The resin was subsequently eluted with 0.8 ml of 98% acetone/2% 0.02 N HCl solution, followed by a second nitrogen flow; the eluate was gathered in the “solution in the reactor”. The columns and the collected samples were subjected to gamma-ray spectrometry analysis. − Labeling and product purification: 10 syntheses were performed in order to investigate 68Zn relative distribution. To this purpose 500 μl of 65Zn in 0.1 N HCl solution, equivalent to an average activity of (17 76) kBq, were loaded on the STRATA X-C column before each synthesis; the activity present in the columns, the wastes and the final product were measured by gamma ray spectrometry. Initial tests allowed to choose the optimal volume of the tracer solution allowing the detection of
G. Lucconi et al. / Applied Radiation and Isotopes 80 (2013) 27–31
both contaminants (65Zn and 68Ge) while keeping the uncertainty in the activity measures reasonably low.
2.4. Gamma ray spectrometry In order to assess 68Ge and 65Zn distribution in the different steps of the synthesis, samples were measured with a high resolution gamma ray spectrometry system (Canberra, distributed in Italy by T.N.E., Milan) after a waiting time of at least 48 h to allow for substantial 68Ga decay. 68Ge has no gamma ray emission but the secular equilibrium condition with 68Ga is reached in about 6 h; the activity of 68Ge at the EOS was thus equal to the activity of 68Ga at the time of measure. The spectrometry system used includes a HPGe detector with a 30% relative efficiency and a resolution of 1.8 keV at 1332 keV; it has been calibrated in the range 59–1836 keV using a multiradionuclide certified reference solution, obtained from an accredited Standardization Laboratory (LEA CERCA, France) and containing 241Am, 109Cd, 57Co, 139Ce, 51Cr, 113Sn, 85Sr, 137Cs, 60Co and 88Y in a mixture made up proportionally to ensure equivalent counts for each energy. The calibration process has been performed according to the IEC 61452 standard (IEC, 1995), using Canberra Genie 2000 software; a dual logarithmic polynomial efficiency curve has been used. The method implemented in the software accounts for propagation of the uncertainties in the calibration of the reference source (1–2% at 1 sigma level, depending on the peak in the mixture), in the tabulated yield (typically o1%), in the net peak area ( o 1% for calibration peaks) and in the interpolation of the efficiency values, the latter being evaluated from the covariance matrix of the fitting of the efficiency curve (typically o3%). The calibration uncertainty thus amounts to about 4–5% at 1 sigma level. Sample spectra were acquired for 16,000 s and analyzed with Canberra Genie 2000 software using a radionuclide library specifically prepared for this study.
29
3. Results The elution yield of 65Zn from 68Ge/68Ga generator resulted (95 75)%; the retention on the resin could be considered negligible as 65Zn activity recovered in the second and third elutions was 3 and 5 orders of magnitude smaller (see Table 1). A direct comparison of the cumulative 65Zn and 68Ga elution profiles is displayed in Fig. 2, showing an overlapping of the two curves within the errors. According to these results, 68Zn stored in the generator is eluted along with 68Ga making the postprocessing of the eluate mandatory in order to reduce the amount of competing 68Zn in the reactor vessel. Tests on the disposable pre-purification STRATA X-C cationic exchange cartridge proved the effectiveness of the chosen purification method since more than 99% of the eluted 65Zn was discarded in the “generator waste”, while only (0.20 70.08)% reached the reactor vessel for the labeling process (Table 2). Consequently, although the amount of 68Zn stored in the 68 Ge/68Ga generator exponentially increases as a function of time from the last elution, even after some days the quantity of this impurity that actually reaches the reactor is negligible compared to the amount of gallium (Fig. 3). Fig. 4 is a typical gamma-ray spectrum of a “generator waste” acquired after a waiting time of at least 48 h from the EOS as to allow for substantial 68Ga decay; the characteristic peak of 65Zn at 1115 keV and the 68Ga emission at 1077 keV (revealing the presence of 68Ge breakthrough) can be
Table 1 65 Zn elution yield from a
68
Ge/68Ga generator.
Elution
65
1 2 3
957 5 (7.2 7 0.5) 10−2 (2.657 0.14) 10−3
Zn elution yield (%)
Fig. 1. Scheme of the fully automated Ga-68-DOTA-conjugated peptides module (Eckert & Ziegler Eurotope).
30
G. Lucconi et al. / Applied Radiation and Isotopes 80 (2013) 27–31
Table 3 65 Zn relative synthesis.
Fig. 2. 65Zn and 68Ga cumulative elution profiles from a (Eckert & Ziegler).
68
Ge/68Ga generator
Table 2 65 Zn relative distribution during the pre-purification process with a STRATA X-C cationic exchange cartridge. Sample
65
Generator waste STRATA X-C column Solution in the reactor
99.727 0.12 0.08 7 0.04 0.20 7 0.08
Zn activity (%)
distribution
in
68
Ga-DOTANOC
Sample
65
Generator waste STRATA X-C column Synthesis waste STRATA X column Product
99.56 7 0.14 0.28 7 0.10 (4.3 7 1.9) 10−3 (3.7 7 1.8) 10−3 0.157 0.05
Zn activity (%)
Table 4 Competition between 68Zn and 68Ga in the synthesis process. 68Ga pmols in the reactor are calculated through Bateman equation considering the generators elution yield; 68Ga pmols in the final solution are obtained from the activity measured with the radioactive activity meter at the EOS; 68Zn pmols are derived from 65Zn activity at the EOS measured with gamma-ray spectrometry. Elution test
Δt from previous elution (h)
reactor
the product
1 2 3 4 5 6 7 8 9 10
20.9 20.8 20.9 19.3 19.2 18.5 20.8 19.5 24.8 116
(2.047 0.12) 10−2 (2.75 7 0.15) 10−2 (2.05 7 0.13) 10−2 (1.79 7 0.11) 10−2 (2.28 7 0.14) 10−2 (1.52 7 0.10) 10−2 (2.23 7 0.14) 10−2 (2.43 7 0.15) 10−2 (4.8 7 0.3) 10−2 (8.7 7 0.5) 10−2
(2.75 7 0.19) 10−2 (3.6 7 0.3) 10−2 (2.8 7 0.2) 10−2 (2.28 7 0.16) 10−2 (3.17 0.2) 10−2 (2.127 0.15) 10−2 (3.4 7 0.2) 10−2 (3.6 7 0.3) 10−2 (7.3 7 0.5) 10−2 (1.22 7 0.09) 10−1
pmolð68 Z nÞ pmolð68 G aÞ
in the
pmolð68 Z n−DOTANOCÞ pmolð68 G a−DOTANOCÞ
in
Table 5 68 Ge breakthrough distribution in 68Ga-DOTANOC synthesis. The activities related to STRATA X column and product samples are MDAs.
68
68
68
68
Fig. 3. Dot lines represent the amount of Zn and Ga eluted from a Ge/ Ga generator as a function of time elapsed from the last elution, obtained from Bateman equations. Solid line represents the amount of 68Zn reaching the reactor vessel, calculated according to the results of Table 2.
Fig. 4. Typical gamma ray spectrum of a “generator waste” sample, acquired with a calibrated HPGe gamma-ray spectrometry system for 16,000 s after a waiting time of at least 48 h from the EOS as to allow for substantial 68Ga decay.
Sample
68
Generator waste STRATA X-C column Synthesis waste STRATA X-C18 column Product
377 4 49 7 5 57 3 3.17 1.1 3.4 7 1.6
Ge EOS activity (%)
easily detected, in addition to the common annihilation peak at 511 and the 40K background radiation. The ability of DOTANOC to bind zinc was evaluated in the whole synthesis process using 65Zn as a radiotracer. The average EOS activity of 68Ga-DOTANOC obtained in the syntheses considered in this study was (1.17 70.07) GBq. (95 72)% of 65Zn present in the reactor vessel formed 65Zn-DOTANOC, co-eluted with 68GaDOTANOC through the disposable STRATA X cartridge. Table 3 displays the average 65Zn distribution in the synthesis process, showing a (0.15 70.05)% of eluted 65Zn in the final solution. The previous outcomes allow to compare the number of moles of 68Zn and 68Ga present in the reactor vessel and in the final product, whose ratio is shown in Table 4 for the 10 syntheses performed. The amount of 68Ga present in the reactor is calculated through Bateman equation considering the elution yield of the two in series generators; the quantity of 68Ga in the final solution is obtained from the activity measured at the EOS; 68Zn pmols are derived from 65Zn activity at the EOS measured with gamma-ray spectrometry. The order of magnitude of this ratio is 10−2, meaning a negligible competition between the radionuclide of interest and its stable daughter in the labeling process. 68 Ge breakthrough relative distribution in each radiosynthesis step is displayed in Table 5, showing that the post-processing of the eluate allows the removal of more than 86% of germanium
G. Lucconi et al. / Applied Radiation and Isotopes 80 (2013) 27–31
Fig. 5. Typical gamma ray spectrum of a “product” sample, acquired with a calibrated HPGe gamma-ray spectrometry system for 16,000 s after a waiting time of at least 48 h from the EOS as to allow for substantial 68Ga decay.
breakthrough. These results, in association with the purification through the STRATA X cartridge, leads to an amount of 68Ge in the final solution inferior to the Minimum Detectable Activity of the HpGe spectrometry system, calculated according to Currie criteria with a confidence level of two sigma (average MDA: (22 79) Bq). Fig. 5 is a typical spectrum of a “product” sample clearly showing the absence of 68Ga peaks. The average 68Ga-DOTANOC radionuclidic purity of the final solution resulted higher than (99.9999986 70.0000006)%, corresponding to a 68Ge content inferior to 1.36 10−6%. 4. Conclusion In this paper we have studied the efficiency of the purification method adopted in one of the most widespread fully automated modules for the synthesis of 68Ga-DOTANOC radiopharmaceutical. The distribution of the two main impurities, 68Ge and 68Zn, has been investigated through radiometric measures available in most PET centers using 65Zn as a radiotracer of stable 68Zn. The results concerning 65Zn elution yield and profile from a 68 Ge/68Ga generator underline the importance of a postprocessing of the eluate granting the separation of 68Ga from the large amount of co-eluted 68Zn; this need is further stressed by the high labeling yield of 68Zn to DOTANOC measured in this work. Among the different purification methods proposed and discussed in the literature (Zhernosekov et al., 2007), cation exchange chromatography seems to be more effective than fractionation in
31
terms of 68Zn purification since the volume of the elution profile containing most of 68Ga, also holds the major part of 68Zn. Our results established that the double purification with STRATA X-C and STRATA X cartridges not only removes more than 96% of 68Ge breakthrough therefore providing a radionuclidic purity superior to 99.9% required by the Pharmacopoeia Monograph on 68Ga Edotreotide injection, but also lowers the competition between 68 Zn and 68Ga in the labeling process which potentially decreases 68 Ga-DOTANOC specific activity. Furthermore, the efficient purification from 68Zn minimizes the need for daily elution of the generators. This efficient purification method leads to an increased pharmaceutical quality and safety of the radiopharmaceutical; moreover highly purified 68Ga fraction could potentially contribute to increase 68Ga-DOTANOC specific activity by lowering the amount of DOTA peptide. Tests are currently on going in order to find the amount of DOTANOC leading to the best tradeoff between labeling yield and specific activity.
References Ambrosini, V., Campana, D., Bodei, L., Nanni, C., Castellucci, P., Allegri, V., Montini, G.C., Tomassetti, P., Paganelli, G., Fanti, S., 2010. 68Ga-DOTANOC PET/CT clinical impact in patients with neuroendocrine tumors. J. Nucl. Med. 51 (5), 669–673. Asti, M., De Pietri, G., Fraternali, A., Grassi, E., Sghedoni, R., Fioroni, F., Roesch, F., Versari, A., Salvo, D., 2008. Validation of 68Ge/68Ga generator processing by chemical purification for routine clinical application of 68Ga-DOTATOC. Nucl. Med. Biol. 35, 721–724. Breeman, W.A.P., Marion, J., Blois, E., Bernard, B.F., Konijnenberg, M., Krenning, E.P., 2005. Radiolabelling DOTA-peptides with 68Ga. Eur. J. Nucl. Med. Mol. Imaging 33, 478–485. Fani, M., Andrè, J.P., Maecke, H.R., 2008. 68Ga-PET: a powerful generator-based alternative to cyclotron-based PET radiopharmaceuticals. Contrast Media Mol. Imaging 3, 67–77. Finn, R., Schlyer, D., 2001. Production considerations for classical PET nuclides. Brookhaven National Lab., Upton, NY (US). IEC, 1995. Measurement of Gamma-Ray Emission Rates of Radionuclides—Calibration and Use of Germanium Spectrometers; IEC 61452, Geneva. Lucconi, G., Cicoria, G., Pancaldi, D., Malizia, C., Marengo, M., 2012. Effective production of 65Zn with a PET cyclotron. Appl. Radiat. Isot. 70 (8), 1590–1594. Lund Nuclear Data Service, 2007. Lund University (Sweden). Fysiska Institutionen, LU, Sölvegatan 14, 223 62 Lund. Available from: 〈http://nucleardata.nuclear.lu. se/nucleardata/toi/〉 (accessed October 2010–March 2011). Ocak, M., Antretter, M., Knopp, R., Kunkel, F., Petrik, M., Bergisadi, N., Decristoforo, C., 2010. Full automation of (68)Ga labelling of DOTA-peptides including cation exchange prepurification. Appl. Radiat. Isot. 68, 297–302. Roesch, F., Riss, P., 2010. The renaissance of the 68Ge/68Ga radionuclide generator initiates new developments in 68Ga radiopharmaceutical chemistry. Curr. Top. Med. Chem. 10 (15). Zhernosekov, K.P., Filosofov, D.V., Baum, R.P., Aschoff, P., Bihl, H., Razbash, A.A., Jahn, M., Jennewein, M., Rösch, F., 2007. Processing of generator-produced 68Ga for medical application. J. Nucl. Med. 48, 1741–1748.
Contents lists available at SciVerse ScienceDirect
Applied Radiation and Isotopes journal homepage: www.elsevier.com/locate/apradiso
Use of 65Zn as a tracer for the assessment of purification in the 68Ga-DOTANOC synthesis G. Lucconi a,b, G. Cicoria b, D. Pancaldi b, F. Lodi c, C. Malizia c, S. Fanti c, S. Boschi c, M. Marengo b,n a
Postgraduate School in Medical Physics, University of Bologna, Italy Medical Physics Department, S.Orsola-Malpighi University Hospital, Bologna, Italy c Nuclear Medicine Unit, S.Orsola-Malpighi University Hospital, Bologna, Italy b
H I G H L I G H T S
We studied 68Ga-DOTANOC radionuclidic purity and 68Zn distribution in the synthesis. 68Zn behavior was inspected by using cyclotron produced 65Zn as a radiotracer. 68Zn competes with 68Ga in labeling DOTANOC with a (95 7 2)% labeling yield. Effectiveness of the STRATA X-C cationic cartridge in lowering the amount of 68Zn to less than 1%. 68Ga-DOTANOC radionuclidic purity was (99.9999986 70.0000006)%, superior to required 99.9%.
art ic l e i nf o
a b s t r a c t
Article history: Received 31 August 2012 Received in revised form 17 April 2013 Accepted 2 May 2013 Available online 30 May 2013
In the last years 68Ga has got into the focus of researchers and clinicians especially for radio-labeling of biomolecules; an important characteristic of this positron emitting isotope is its availability via the 68 Ge/68Ga generator system: the long-lived 68Ge (t1/2 ¼270.8 d) produces the short-lived 68Ga (t1/2 ¼ 67.63 min) which decays to stable 68Zn. 68Ge breakthrough compromises 68Ga radionuclidic purity, while 68 Zn might affect the specific activity of the radiopharmaceutical. In this paper we investigated the weight of these impurities in 68Ga-DOTANOC synthesis. 65 Zn (t1/2 ¼ 244.26 d; decay mode: EC 98.3%, β+ 1.7%) was used as a radiotracer of stable 68Zn; samples of the purification columns, wastes and product were recovered and measured with a calibrated HPGe gamma-ray spectrometry system. The results showed that 68Zn competes with 68Ga in labeling DOTANOC with a (957 2)% labeling yield; they also proved the effectiveness of the STRATA X-C cationic post-processing of the generator eluate in lowering the amount of this impurity to less than 1%. Moreover this approach, along with the purification of the final product through a STRATA X cartridge, effectively removes 68Ge breakthrough providing a 68Ga-DOTANOC radionuclidic purity of (99.9999986 7 0.0000006)%, superior to 99.9% required by the Pharmacopoeia Monograph on 68Ga Edotreotide injection. & 2013 Elsevier Ltd. All rights reserved.
Keywords: 68 Ga-DOTANOC 65 Zn Radioactive tracer Specific activity Radionuclidic purity
1. Introduction Positron-emission tomography (PET) is a well-established diagnostic imaging technique in nuclear medicine; it combines high sensitivity and relatively high resolution, therefore allowing early biochemical assessment of pathology. The recent development of hybrid PET/CT scans has allowed an integration of functional imaging and anatomical localization at a superior
n
Corresponding author. Tel.: +39 051 6363575; fax: +39 051 6363571. E-mail address: [email protected] (M. Marengo).
0969-8043/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.apradiso.2013.05.006
spatial resolution, consequently changing many fields of medical diagnosis. The most frequently used positron emitters are 18F, 11C, 13N and 15 O, produced in a cyclotron through the irradiation of liquid and gaseous targets (Finn and Schlyer, 2001). 68Ga is a positron emitter of great interest because of its physical half-life which is compatible with the pharmacokinetics of most radiopharmaceuticals of low molecular weight; besides it is available via 68Ge/68Ga generators which avoid the need for an on-site cyclotron (Fani et al., 2008). This radionuclide (t1/2 ¼67.63 min; decay mode: 89% β+, 11% EC; Emax(β+)¼1.90 MeV (Lund Nuclear Data Service, 2007)) is indeed conveniently produced exploiting the secular equilibrium
28
G. Lucconi et al. / Applied Radiation and Isotopes 80 (2013) 27–31
with the parent radionuclide 68Ge (t1/2 ¼ 270.8 d; decay mode: 100% EC (Lund Nuclear Data Service, 2007)), trapped on an inorganic or organic resin (Roesch and Riss, 2010). The long halflife of the latter grants 68Ga availability for long periods of time and allows to elute the generator twice a day. Recently, 68Ga-DOTA labeled peptides have become the gold standard for the evaluation of neuroendocrine tumors (NET) with PET/CT, being superior to conventional imaging modalities and the previous 111In-Octreotide SPECT technique (Ambrosini et al., 2010). Despite the convenient availability, most of the 68Ge/68Ga generator systems are yet not optimally designed for direct radio-labeling because of the low pH and large volume of the eluate (Asti et al., 2008); in addition the solution contains measurable activities of long-lived 68Ge and other metal impurities such as 68Zn produced by 68Ga decay. 68Ge affects the radionuclidic purity of the radiopharmaceutical; stable 68Zn, by competing with 68Ga in the labeling reaction, may compromise its specific activity which should be kept as high as possible in order to maximize the tumour-to-background ratio. The DOTA conjugated peptide DOTANOC binds to 68Ga with a ligand to metal ratio 1:1; since 1 nmol of 68Ga holds an activity of 98 GBq, the theoretical specific activity of 68Ga-DOTANOC is 98 GBq/nmol (Zhernosekov et al., 2007). Yet this value may be lowered by the presence of contaminants competing with 68Ga for incorporation in DOTA, such as Zn2+ and Fe2+/3+ (Breeman et al., 2005). In this study we investigated the efficiency of the cationic postprocessing of the 68Ga-eluate implemented in the Eckert & Ziegler Eurotope module for 68Ga DOTA conjugated peptides synthesis. Particular attention was directed to the investigation of 68Zn relative distribution in the different steps of the synthesis using 65 Zn, produced at our laboratory, as a radiotracer. This choice allowed the study of 68Zn distribution through radiometric measures available in most PET centers, thus avoiding more complex and expensive analysis with mass spectrometer devices.
2. Materials and methods 2.1.
68
Ga production
68 Ga was obtained from two in series 68Ge/68Ga generators (Eckert & Ziegler Eurotope, distributed in Italy by Radius, Budrio, BO), each with an initial activity of 1850 MBq at the calibration date, with a difference of nine months between the two generators. The certified initial elution yield was (76.3 70.9)% and (86 74)% and was supposed to remain greater than 70% for the following nine months; 68Ge breakthrough resulted less than 5 10−3% of the total eluted activity.
2.2.
68
68
Ga-DOTANOC synthesis
Ga-DOTANOC syntheses were performed with the (Eckert & Ziegler) Modular Lab automated system. DOTANOC precursor (ABX GmbH, Radeberg, Germany) and all the other reagents were of pharmaceutical grade. Two in series 68Ge/68Ga generators were eluted with 15 ml of 0.1 N HCl at a flow of 2 ml/min using a remote controlled pump (Fig. 1). The eluate was loaded onto a disposable Phenomenex STRATA X-C cationic exchange cartridge in order to remove 68Ge breakthrough and other metal impurities in the “generator waste”. In a second step the column was washed with 0.8 ml of 98% acetone/2% 0.02 N HCl solution to recover trapped 68Ga, which was directly transferred to the reactor vessel, containing 40 mg of DOTANOC diluted in a 0.2 N sodium acetate/acetic acid buffer, pH
(4.070.2). The solution was heated to 90 1C for 5 min and then loaded onto a pre-conditioned Phenomenex STRATA X reversed phase cartridge for purification from unreacted 68Ga, collected in the “synthesis waste”. 68Ga-DOTANOC was finally recovered with 0.8 ml of ethanol, diluted with 7.0 ml of saline and then sterilized with a 0.22 μm filter (Ocak et al., 2010). The activity of the labeled solution was measured at the End of Synthesis (EOS) with a CRC15 PET ionization chamber radionuclide activity meter (Capintec Inc., Ramsey, NJ) calibrated by means of NIST traceable standard (mod. BM06S-685, RadQual Inc., Weare NH).
2.3.
5
Zn production and application
Despite the large availability of 65Zn solutions, we used 65Zn previously produced at our laboratory via the 65Cu(p,n)65Zn nuclear reaction using a 16.5 MeV GE-PET trace biomedical cyclotron (Lucconi et al., 2012). Natural copper foils (thickness: 100 μm; purity: 499.9%) were irradiated in a solid target station developed in our institution; 65Zn was purified from copper and other long-lived contaminants through a BIO RAD AG1-X8 anionic exchange resin 100–200 mesh Cl− form (Bio-Rad Lab., California, USA) and finally transferred to a 0.1 N HCl solution suitable for radiopharmaceutical synthesis. The radionuclidic purity of the tracer, expressed in activity, was 4(99.921 70.003)%. 65 Zn solution was used as a tracer of 68Zn in different steps of the synthesis: − The elution yield of 68Zn from a 68Ge/68Ga generator: a single Eckert & Ziegler 68Ge/68Ga decommissioned generator was eluted with 1 ml of 65Zn solution holding an activity of (25.3 71.0) kBq, followed by 9 ml of 0.1 N HCl in order to simulate standard elution volume (10 ml of 0.1 N HCl). The generator was then regularly eluted twice as to study the eventual retention of 65Zn on the resin. The eluates were investigated through gamma-ray spectrometry. − The elution profile of 68Zn from a 68Ge/68Ga generator: the death volume of the same decommissioned generator was measured with 0.1 N HCl; 1 ml of 65Zn solution was then loaded followed by enough air to place the tracer in the middle of the death volume, where the resin was supposed to be. The generator was eluted with 15 ml of 0.1 N HCl and the elution profile was sampled with 1 cm3 tubes. 68Ga activity was measured with a CRC15 PET ionization chamber radionuclide activity meter (Capintec Inc., Ramsey, NJ), while 65Zn activity was measured with gamma ray spectrometry after substantial 68Ga decay in order to improve the accuracy of the measurement. − Purification with the STRATA X-C cationic exchange cartridge: 5 tests were performed in order to investigate the low affinity of the resin with zinc. 1 ml of 65Zn solution was loaded onto a STRATA X-C column in standard synthesis conditions. The column was then eluted with 9 ml of 0.1 N HCl and taken to dryness with a 30 s nitrogen flow, while collecting the solution in the “generator waste”. The resin was subsequently eluted with 0.8 ml of 98% acetone/2% 0.02 N HCl solution, followed by a second nitrogen flow; the eluate was gathered in the “solution in the reactor”. The columns and the collected samples were subjected to gamma-ray spectrometry analysis. − Labeling and product purification: 10 syntheses were performed in order to investigate 68Zn relative distribution. To this purpose 500 μl of 65Zn in 0.1 N HCl solution, equivalent to an average activity of (17 76) kBq, were loaded on the STRATA X-C column before each synthesis; the activity present in the columns, the wastes and the final product were measured by gamma ray spectrometry. Initial tests allowed to choose the optimal volume of the tracer solution allowing the detection of
G. Lucconi et al. / Applied Radiation and Isotopes 80 (2013) 27–31
both contaminants (65Zn and 68Ge) while keeping the uncertainty in the activity measures reasonably low.
2.4. Gamma ray spectrometry In order to assess 68Ge and 65Zn distribution in the different steps of the synthesis, samples were measured with a high resolution gamma ray spectrometry system (Canberra, distributed in Italy by T.N.E., Milan) after a waiting time of at least 48 h to allow for substantial 68Ga decay. 68Ge has no gamma ray emission but the secular equilibrium condition with 68Ga is reached in about 6 h; the activity of 68Ge at the EOS was thus equal to the activity of 68Ga at the time of measure. The spectrometry system used includes a HPGe detector with a 30% relative efficiency and a resolution of 1.8 keV at 1332 keV; it has been calibrated in the range 59–1836 keV using a multiradionuclide certified reference solution, obtained from an accredited Standardization Laboratory (LEA CERCA, France) and containing 241Am, 109Cd, 57Co, 139Ce, 51Cr, 113Sn, 85Sr, 137Cs, 60Co and 88Y in a mixture made up proportionally to ensure equivalent counts for each energy. The calibration process has been performed according to the IEC 61452 standard (IEC, 1995), using Canberra Genie 2000 software; a dual logarithmic polynomial efficiency curve has been used. The method implemented in the software accounts for propagation of the uncertainties in the calibration of the reference source (1–2% at 1 sigma level, depending on the peak in the mixture), in the tabulated yield (typically o1%), in the net peak area ( o 1% for calibration peaks) and in the interpolation of the efficiency values, the latter being evaluated from the covariance matrix of the fitting of the efficiency curve (typically o3%). The calibration uncertainty thus amounts to about 4–5% at 1 sigma level. Sample spectra were acquired for 16,000 s and analyzed with Canberra Genie 2000 software using a radionuclide library specifically prepared for this study.
29
3. Results The elution yield of 65Zn from 68Ge/68Ga generator resulted (95 75)%; the retention on the resin could be considered negligible as 65Zn activity recovered in the second and third elutions was 3 and 5 orders of magnitude smaller (see Table 1). A direct comparison of the cumulative 65Zn and 68Ga elution profiles is displayed in Fig. 2, showing an overlapping of the two curves within the errors. According to these results, 68Zn stored in the generator is eluted along with 68Ga making the postprocessing of the eluate mandatory in order to reduce the amount of competing 68Zn in the reactor vessel. Tests on the disposable pre-purification STRATA X-C cationic exchange cartridge proved the effectiveness of the chosen purification method since more than 99% of the eluted 65Zn was discarded in the “generator waste”, while only (0.20 70.08)% reached the reactor vessel for the labeling process (Table 2). Consequently, although the amount of 68Zn stored in the 68 Ge/68Ga generator exponentially increases as a function of time from the last elution, even after some days the quantity of this impurity that actually reaches the reactor is negligible compared to the amount of gallium (Fig. 3). Fig. 4 is a typical gamma-ray spectrum of a “generator waste” acquired after a waiting time of at least 48 h from the EOS as to allow for substantial 68Ga decay; the characteristic peak of 65Zn at 1115 keV and the 68Ga emission at 1077 keV (revealing the presence of 68Ge breakthrough) can be
Table 1 65 Zn elution yield from a
68
Ge/68Ga generator.
Elution
65
1 2 3
957 5 (7.2 7 0.5) 10−2 (2.657 0.14) 10−3
Zn elution yield (%)
Fig. 1. Scheme of the fully automated Ga-68-DOTA-conjugated peptides module (Eckert & Ziegler Eurotope).
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G. Lucconi et al. / Applied Radiation and Isotopes 80 (2013) 27–31
Table 3 65 Zn relative synthesis.
Fig. 2. 65Zn and 68Ga cumulative elution profiles from a (Eckert & Ziegler).
68
Ge/68Ga generator
Table 2 65 Zn relative distribution during the pre-purification process with a STRATA X-C cationic exchange cartridge. Sample
65
Generator waste STRATA X-C column Solution in the reactor
99.727 0.12 0.08 7 0.04 0.20 7 0.08
Zn activity (%)
distribution
in
68
Ga-DOTANOC
Sample
65
Generator waste STRATA X-C column Synthesis waste STRATA X column Product
99.56 7 0.14 0.28 7 0.10 (4.3 7 1.9) 10−3 (3.7 7 1.8) 10−3 0.157 0.05
Zn activity (%)
Table 4 Competition between 68Zn and 68Ga in the synthesis process. 68Ga pmols in the reactor are calculated through Bateman equation considering the generators elution yield; 68Ga pmols in the final solution are obtained from the activity measured with the radioactive activity meter at the EOS; 68Zn pmols are derived from 65Zn activity at the EOS measured with gamma-ray spectrometry. Elution test
Δt from previous elution (h)
reactor
the product
1 2 3 4 5 6 7 8 9 10
20.9 20.8 20.9 19.3 19.2 18.5 20.8 19.5 24.8 116
(2.047 0.12) 10−2 (2.75 7 0.15) 10−2 (2.05 7 0.13) 10−2 (1.79 7 0.11) 10−2 (2.28 7 0.14) 10−2 (1.52 7 0.10) 10−2 (2.23 7 0.14) 10−2 (2.43 7 0.15) 10−2 (4.8 7 0.3) 10−2 (8.7 7 0.5) 10−2
(2.75 7 0.19) 10−2 (3.6 7 0.3) 10−2 (2.8 7 0.2) 10−2 (2.28 7 0.16) 10−2 (3.17 0.2) 10−2 (2.127 0.15) 10−2 (3.4 7 0.2) 10−2 (3.6 7 0.3) 10−2 (7.3 7 0.5) 10−2 (1.22 7 0.09) 10−1
pmolð68 Z nÞ pmolð68 G aÞ
in the
pmolð68 Z n−DOTANOCÞ pmolð68 G a−DOTANOCÞ
in
Table 5 68 Ge breakthrough distribution in 68Ga-DOTANOC synthesis. The activities related to STRATA X column and product samples are MDAs.
68
68
68
68
Fig. 3. Dot lines represent the amount of Zn and Ga eluted from a Ge/ Ga generator as a function of time elapsed from the last elution, obtained from Bateman equations. Solid line represents the amount of 68Zn reaching the reactor vessel, calculated according to the results of Table 2.
Fig. 4. Typical gamma ray spectrum of a “generator waste” sample, acquired with a calibrated HPGe gamma-ray spectrometry system for 16,000 s after a waiting time of at least 48 h from the EOS as to allow for substantial 68Ga decay.
Sample
68
Generator waste STRATA X-C column Synthesis waste STRATA X-C18 column Product
377 4 49 7 5 57 3 3.17 1.1 3.4 7 1.6
Ge EOS activity (%)
easily detected, in addition to the common annihilation peak at 511 and the 40K background radiation. The ability of DOTANOC to bind zinc was evaluated in the whole synthesis process using 65Zn as a radiotracer. The average EOS activity of 68Ga-DOTANOC obtained in the syntheses considered in this study was (1.17 70.07) GBq. (95 72)% of 65Zn present in the reactor vessel formed 65Zn-DOTANOC, co-eluted with 68GaDOTANOC through the disposable STRATA X cartridge. Table 3 displays the average 65Zn distribution in the synthesis process, showing a (0.15 70.05)% of eluted 65Zn in the final solution. The previous outcomes allow to compare the number of moles of 68Zn and 68Ga present in the reactor vessel and in the final product, whose ratio is shown in Table 4 for the 10 syntheses performed. The amount of 68Ga present in the reactor is calculated through Bateman equation considering the elution yield of the two in series generators; the quantity of 68Ga in the final solution is obtained from the activity measured at the EOS; 68Zn pmols are derived from 65Zn activity at the EOS measured with gamma-ray spectrometry. The order of magnitude of this ratio is 10−2, meaning a negligible competition between the radionuclide of interest and its stable daughter in the labeling process. 68 Ge breakthrough relative distribution in each radiosynthesis step is displayed in Table 5, showing that the post-processing of the eluate allows the removal of more than 86% of germanium
G. Lucconi et al. / Applied Radiation and Isotopes 80 (2013) 27–31
Fig. 5. Typical gamma ray spectrum of a “product” sample, acquired with a calibrated HPGe gamma-ray spectrometry system for 16,000 s after a waiting time of at least 48 h from the EOS as to allow for substantial 68Ga decay.
breakthrough. These results, in association with the purification through the STRATA X cartridge, leads to an amount of 68Ge in the final solution inferior to the Minimum Detectable Activity of the HpGe spectrometry system, calculated according to Currie criteria with a confidence level of two sigma (average MDA: (22 79) Bq). Fig. 5 is a typical spectrum of a “product” sample clearly showing the absence of 68Ga peaks. The average 68Ga-DOTANOC radionuclidic purity of the final solution resulted higher than (99.9999986 70.0000006)%, corresponding to a 68Ge content inferior to 1.36 10−6%. 4. Conclusion In this paper we have studied the efficiency of the purification method adopted in one of the most widespread fully automated modules for the synthesis of 68Ga-DOTANOC radiopharmaceutical. The distribution of the two main impurities, 68Ge and 68Zn, has been investigated through radiometric measures available in most PET centers using 65Zn as a radiotracer of stable 68Zn. The results concerning 65Zn elution yield and profile from a 68 Ge/68Ga generator underline the importance of a postprocessing of the eluate granting the separation of 68Ga from the large amount of co-eluted 68Zn; this need is further stressed by the high labeling yield of 68Zn to DOTANOC measured in this work. Among the different purification methods proposed and discussed in the literature (Zhernosekov et al., 2007), cation exchange chromatography seems to be more effective than fractionation in
31
terms of 68Zn purification since the volume of the elution profile containing most of 68Ga, also holds the major part of 68Zn. Our results established that the double purification with STRATA X-C and STRATA X cartridges not only removes more than 96% of 68Ge breakthrough therefore providing a radionuclidic purity superior to 99.9% required by the Pharmacopoeia Monograph on 68Ga Edotreotide injection, but also lowers the competition between 68 Zn and 68Ga in the labeling process which potentially decreases 68 Ga-DOTANOC specific activity. Furthermore, the efficient purification from 68Zn minimizes the need for daily elution of the generators. This efficient purification method leads to an increased pharmaceutical quality and safety of the radiopharmaceutical; moreover highly purified 68Ga fraction could potentially contribute to increase 68Ga-DOTANOC specific activity by lowering the amount of DOTA peptide. Tests are currently on going in order to find the amount of DOTANOC leading to the best tradeoff between labeling yield and specific activity.
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