Simplified NaCl Based 68Ga Concentration and Labeling Procedure

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Simplified NaCl Based 68Ga Concentration and Labeling Procedure for Rapid Synthesis of 68Ga Radiopharmaceuticals in High Radiochemical Purity Dirk Mueller,*,† Ingo Klette,† Richard P. Baum,† M. Gottschaldt,‡ Michael K. Schultz,¶ and Wouter A. P. Breeman§ †

Zentralklinik Bad Berka, Department of Nuclear Medicine/PET Center, 99437 Bad Berka, Germany Friedrich Schiller University of Jena, Institute for Organic and Macromolecular Chemistry, Jena Center for Soft Matter (JCSM), 07743 Jena, Germany ¶ University of Iowa, Departments of Radiology and Radiation Oncology (Free Radical Radiation Biology Program), ML B180, 500 Newton Road, Iowa City, Iowa 52242, United States § Department of Nuclear Medicine, Erasmus MC Rotterdam, ’s Gravendijkwal 230, 3015CE Rotterdam, The Netherlands ‡

ABSTRACT: A simple sodium chloride (NaCl) based 68Ga eluate concentration and labeling method that enables rapid, high-efficiency labeling of DOTA conjugated peptides in high radiochemical purity is described. The method utilizes relatively few reagents and comprises minimal procedural steps. It is particularly well-suited for routine automated synthesis of clinical radiopharmaceuticals. For the 68Ga generator eluate concentration step, commercially available cation-exchange cartridges and 68Ga generators were used. The 68 Ga generator eluate was collected by use of a strong cation exchange cartridge. 98% of the total activity of 68Ga was then eluted from the cation exchange cartridge with 0.5 mL of 5 M NaCl solution containing a small amount of 5.5 M HCl. After buffering with ammonium acetate, the eluate was used directly for radiolabeling of DOTATOC and DOTATATE. The 68Ga-labeled peptides were obtained in higher radiochemical purity compared to other commonly used procedures, with radiochemical yields greater than 80%. The presence of 68Ge could not be detected in the final product. The new method obviates the need for organic solvents, which eliminates the required quality control of the final product by gas chromatography, thereby reducing postsynthesis analytical effort significantly. The 68Ga-labeled products were used directly, with no subsequent purification steps, such as solid-phase extraction. The NaCl method was further evaluated using an automated fluid handling system and it routinely facilitates radiochemical yields in excess of 65% in less than 15 min, with radiochemical purity consistently greater than 99% for the preparation of 68Ga-DOTATOC.

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INTRODUCTION PET imaging with 68Ga-labeled compounds is of increasing interest in nuclear medicine worldwide. 68Ga-labeled DOTApeptides, such as DOTA-conjugated somatostatin analogues DOTATOC and DOTATATE, are particularly well suited for PET imaging of tumors expressing somatostatin receptors.1−3 The synthesis of 68Ga-based radiopharmaceuticals, in principle, can be performed on site, without the need for a medical cyclotron.4 However, widespread use of 68Ga-based radiopharmaceuticals in clinical practice has been limited due to concerns related to parent radionuclide 68Ge breakthrough, concentration and purification of 68Ga in advance of the radiolabeling step, and analytical requirements for quality control.5 As generator technologies have matured to more consistently deliver highpurity 68Ga over many months, the availability of highly adaptable postprocessing radiochemistry modules has also increased. These developments suggest a rich future for generator-produced 68Ga as an attractive candidate for radiolabeling of not only peptides, but also other molecular targeting © 2012 American Chemical Society

vectors such as bisphosphonates, amino acids, or carbohydrates.6−9 Although numerous methods for the elution of 68Ga from the generator, its purification, and for labeling peptide radiopharmaceuticals with it have been advanced, these approaches are primarily based on four different 68Ga eluate concentration and purification procedures (Figure 1). Meyer et al. described a method for 68Ga concentration and purification via an anion exchange column (Figure 1, route 1).10 In this procedure, 68Ga chloride from the 68Ge/68Ga generator eluate is converted into the gallate anion [68GaCl4]− by addition of concentrated HCl (final [Cl−] > 5 M). [68GaCl4]− is then trapped on an anionic exchange cartridge and is subsequently eluted with a small volume of water into a buffer solution (containing, e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic Received: March 11, 2012 Revised: June 25, 2012 Published: July 4, 2012 1712

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Technical Note

Figure 1. Schematic drawing of the currently mostly applied 68Ge/68Ga-generator eluate concentration methods prior to 68Ga labeling reactions: SCX, strong cation exchanger; SAX, strong anion exchanger; 1, anionic eluate concentration; 2, combined cationic/anionic eluate concentration; 3, fractionated method; 4, cationic eluate concentration.

Zhernosekov et al.,14 which is based on a two-step approach. 68Ga eluted from the generator in dilute HCl is trapped on a cation exchange resin, washed and eluted with two different hydrochloric acid/acetone solutions (Figure 1, route 4). The majority of acetone is removed during the radiolabeling step, which is carried out at 100 °C. To ensure that the final product is of sufficient radiochemical purity (>95%), the procedure includes a purification step using a solid-phase extraction. Although this method has proven to be effective and reliable, the disadvantage for routine clinical production is the use of organic solvents acetone and ethanol. The use of these organic solvents requires additional quality control testing of the final products that not only adds to the production effort for technical staff, but also requires purchase and maintenance of additional quality control instrumentation (e.g., gas chromatography equipment). Furthermore, the acetone-HCl-based solution may contain breakdown impurities (mesityloxide, 4-methyl-3-penten-2-on), which can be detected in the final products. Although these methods have been proven to deliver 68Ga-labeled compounds in high radiochemical purity and specific activity, the increasing number of 68Ga labeling procedures performed in our facilities prompted us to explore more rapid methods, with reduced quality control requirements and in which organic solvents could be removed from the preparation protocol. Thus, in this work, we report a new NaCl-based highly efficient 68Ga eluate concentration and labeling procedure for DOTA conjugated peptides that is particularly well-suited for routine production and clinical use and has the potential to be expanded to other ligands.

acid, HEPES) for the labeling step. After labeling, further purification steps are necessary in order to remove HEPES. In addition, 68Ge that might still be present is removed by solidphase extraction, while the final product is eluted with ethanol. A second approach for the concentration of 68Ga takes advantage of the occurrence of different gallium species (Ga3+ cations and anionic [GaCl4]−) depending on varying pH and chloride concentrations. In this procedure, 68Ga is trapped on a strong cation exchange cartridge (SCX) and then converted to [68GaCl4]− and eluted with a small volume of 5.5 M HCl. In step two, the [68GaCl4]− is adsorbed on a strong anion exchange cartridge (SAX) and 68Ga3+ is subsequently eluted with water (Figure 1, route 2). This concentration method is particularly well suited for the subsequent labeling of fragile peptides such as DOTA conjugated Affibody molecules (e.g., DOTAZHer2:342‑pep2).11,12 Because of the high purity of the labeled peptide after the radiolabeling reaction step, a final purification step is usually not required. However, the method requires two cartridges. A third approach to obtain 68Ga-labeled peptides uses the generator eluate directly by a fractional elution of the 68Ge/68Gagenerator. In this method, the fraction with the highest volume activity is collected and buffered to an appropriate pH for radiolabeling (e.g., in HEPES).13 While potentially effective, this method has the drawback that only a fraction of the eluted 68Ga activity is used, which potentially reduces the achievable specific activity of the final product (Figure 1, route 3). The fourth and most popular methodology currently used for the synthesis of 68Ga-labeled compounds was described by 1713

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EXPERIMENTAL SECTION

Technical Note

RESULTS AND DISCUSSION The foundation for our initiatives is based on the supposition that 68 Ga adsorbed onto a silica-based SCX cation exchange cartridge should be elutable with a concentrated NaCl solution by conversion of adsorbed 68Ga3+ to the [68GaCl4]− anion in the presence of a high concentration of chloride anions. In our initial experiments, it was found that 70% of the adsorbed 68Ga activity can be eluted with 3 mL of 5 M NaCl solution from a SCX cartridge, which was preloaded with 68Ga directly from the generator 0.1 M HCl eluate. Indeed, the eluate from the SCX column was examined by radio HPLC and the main peak at 2.2 min corresponds to the retention time for [68GaCl4]− established previously (Figure 2).11,12,15 Due to the large elution volume, the

All reagents were purchased from commercial sources and used as received. Cartridges used for separation are commercially available from Varian. For all experiments, a 68Ga generator from Obninsk (Eckert & Ziegler Europe) or an IGG100 68Ga generator (Eckert & Ziegler Europe) were used. The total radioactivity used for the labeling reactions was between 1.6 and 2.0 GBq over a period of two months. The SCX cartridge (Varian, Bond Elut-SCX, 100 mg, 1 mL) was preconditioned with 1 mL 5.5 M HCl and 10 mL water prior to the elution step. For this investigation, the radiochemical purity was determined by HPLC using the following conditions: HPLC pump, Jasco PU-1580; quaternary gradient unit, Jasco LG-1580−04; radio detector, biostep IsoScan LC; multiwavelength detector, Jasco MD 1510; column, RP-18, LiChroCART 250−4, LiChrospher 100, RP-18 (5 μm). Radiochemical purity was also examined by routine radio-thin layer chromatography using ITLC-SG (Varian) (mobile phase: acetonitrile/water 1:1). The radiochemical yields are expressed as a percentage of total activity of 68 Ga from the generator eluates and are not decay-corrected. Labeling of DOTA Conjugated Peptides Using the NaCl Based Method. The 68Ga generators were eluted with a total of 10 mL of 0.1 M HCl. On a SCX cartridge, 99.9% of 68Ga of the generator eluate was collected and subsequently eluted with minimal loss (1−2%) using a mixture of 12.5 μL of 5.5 M HCl and 500 μL of 5 M NaCl. This eluate was slowly added to a solution of 200−400 μL ammonium acetate buffer (1 M) and 40 μg of the DOTA conjugated peptide (DOTATOC or DOTATATE) in 3.0 mL of water. The acetate buffer solution was prepared using a mixture of 3.9 g ammonium acetate and 1 mL conc. HCl, diluted to 50 mL with water, and the pH was adjusted with glacial acetic acid to 4.5. When mixed, the final pH of the radiolabeling reaction mixture was determined to be 3.6 ± 0.3. The radiolabeling reaction was carried out at 90 °C for 7 min. Finally, the reaction mixture was neutralized with 2 mL of sterile sodium phosphate buffer (1 mmol/L Na+, 0.6 mmol/L PO43‑, pH = 7.0; B. Braun, Melsungen AG, Germany) and can be diluted further for dose administration to any desired volume. Using this approach, radiochemical purity of >99% was routinely achieved for the labeled peptide as determined by radio HPLC. The radiochemical yield after sterile filtration was 80% ± 2% (manual method). Automated Concentration and Labeling Procedure. DOTATOC was labeled on a Modular-Lab PharmTracer module (Eckert & Ziegler). The elution of the 68Ga generators, the 68Ga concentration step, and the subsequent elution of 68Ga using NaCl/HCl were carried out similarly as described above. This eluate was slowly added into a solution of 350 μL ammonium acetate buffer (as mentioned above), 5 mg ascorbic acid, and 50 μg of the DOTA conjugated peptide (DOTATOC) in 3.0 mL of water. The final pH of the reaction mixture was determined to be 3.6 ± 0.3. After heating the solution (7 min at 90 °C), the 68Ga chloride is quantitatively coupled to the DOTA moiety of the peptide conjugate. The reaction mixture was diluted with 2 mL of water and sterile-filtered. Radiochemical purity of >99% is routinely achieved as determined by radio HPLC. The radiochemical yield after sterile filtration was about 65%, which is comparable to the manual method using acetone/ HCl. The final product was neutralized with 2 mL of sterile sodium phosphate buffer (1 mmol/L Na+, 0.6 mmol/L PO43−; B. Braun, Melsungen AG, Germany). The total duration of the automated synthesis was 14 min.

Figure 2. HPLC chromatogram of the SCX cartridge eluate in 5 M NaCl. HPLC: column: RP-18, LiChroCART 250−4, LiChrospher 100, RP-18 (5 μm); solvent A: acetonitrile solution in water (5%), 0.1% TFA; solvent B: 95% acetonitrile solution in water, 0.1% TFA; flow rate: 1.2 mL/min; gradient: from 0 to 2 min 100% A, 2−15 min to 100% B; main peak of [68GaCl4]− at 2.2 min.

obtained [68GaCl4]− had to be trapped on a SAX cartridge, but after elution with water, it could be directly used for the labeling of DOTATOC. Later, we found that, by addition of a minimum amount of HCl to the 5 M NaCl solution, the 68Ga activity was almost quantitatively elutable directly from the SCX cartridge. In this way, the amount of HCl could be reduced in the final elution step. The optimal mixture was found to be 12.5 μL of 5.5 M HCl in 0.5 mL of 5 M NaCl solution. This eluate could be used directly for 68Ga labeling of DOTA conjugated peptides in buffered reaction mixtures. This new NaCl-based concentration and labeling procedure is a combination of a cationic trapping and an intermediate transformation of the absorbed 68Ga3+ into [68GaCl4]− by elution with 5 M NaCl solution and re-transformation to 68Ga3+ with the help of acetate buffer. Figure 3 shows the scheme of the complete procedure for the labeling of DOTATOC with 68Ga. It was found that ammonium acetate is suited for buffering the reaction solution to an optimal pH between 3 and 4.16 The radiochemical and chemical purity of the sterile-filtered final product allows for direct use of the neutralized radiopharmaceutical without a subsequent purification step to remove unreacted 68Ga. If ascorbic acid (5 mg) was added to the peptide/ buffer solution before the labeling reaction, the 68 GaDOTATOC is stable for more than two half-lives. As shown in Figure 4A, radiolysis was not detectable by radio HPLC, even two hours after the labeling reaction. In contrast, even after a final purification step, the 68Ga-labeled DOTATOC produced by the 1714

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Technical Note

Figure 3. Schematic drawing of the NaCl-based labeling procedure.

acetone/HCl concentration procedure14 had side products that can be detected by radio HPLC (Figure 4B). For the labeling of DOTATATE using the HCl/acetone procedure, larger amounts of side products are frequently seen in the final solution most probably arising from unspecific partial hydrolysis or hydroxylation by radiolysis of the peptide residue (Figure 5B). A subsequent purification with the help of a solidphase extraction would only separate the free 68Ga. In these cases, the radiochemical purity of the final product can be lower than 90% and therefore will not be sufficient for clinical use. In contrast, the NaCl method described herein delivers the 68GaDOTATATE in high radiochemical purity (with no apparent side products) as detected by radio HPLC (Figure 5A). To further evaluate the new NaCl method for routine radiopharmaceutical production, the manual preparation was adapted for use with an automated fluid-handling system for labeling of DOTATOC (Table 1). For these experiments, a Modular-Lab PharmTracer automated fluid handling module (Eckert & Ziegler) was employed. The optimal radiolabeling conditions were found to be the use of 250−450 μL 1 M ammonium acetate buffer and 30−50 μg of DOTATOC with 3.0 mL of water. Additionally, by using the NaCl based method, the synthesis time could be significantly reduced (14 min) compared to the HCl/acetone method, thereby improving overall radiochemical yields (a radiochemical yield of 65% could be achieved). After complete decay of 68Ga (t1/2 = 68 min), analysis by highpurity germanium detector revealed no evidence of parent radionuclide 68Ge in the final products (i.e., no significant difference between background and sample spectrum), whether performed manually or by automated spectral analysis methods. The main 68Ge breakthrough from the generator was not

Figure 4. (A) Typical HPLC chromatogram of 68Ga-DOTATOC using the NaCl-based labeling procedure, 2 h after synthesis. (B) Typical HPLC chromatogram of purified 68Ga-DOTATOC using the acetone/ HCl method immediately after synthesis; for HPLC conditions, see Figure 2.

adsorbed on the SCX cartridge during the initial adsorption of Ga and is therefore found in the SCX waste. A small amount of 68 Ge activity was retained on the cartridge, but was not eluted with the NaCl/HCl solution. These results are consistent with other published methods.13 With the success in adapting the new NaCl method for automated preparation of radiopharmaceuticals, we have used this preparation routinely in more than 300 automated runs for the production of 68Ga-DOTATOC, which were applied for molecular imaging of more than 800 patients. 68

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CONCLUSION The use of 5 M NaCl solution containing a very low amount of HCl in the initial step for 68Ga concentration leads to a greatly simplified concentration and labeling procedure for DOTA conjugated peptides. The one-step concentration method provides >98% of generator-based 68Ga eluant for radiolabeling, and can be used directly for the radiolabeling reaction. A radiochemical purity greater than 99% was routinely achieved. Compared to the widely used acetone/HCl procedure, no peptide side products were observed by radio HPLC. In addition, 1715

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patient studies. The method was adapted to an automated synthesis module leading to a rapid preparation (14 min vs 32 min in our hands). Therefore, the described procedure makes 68 Ga-labeled compounds more easily accessible for institutions with less frequent production needs (and smaller quality control instrumentation budgets), as well as for routine preparations in centers with heavy production requirements via reduced analytical effort and more rapid production.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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(1) Prasad, V., and Baum, R. P. (2010) Biodistribution of the Ga-68 labeled somatostatin analogue DOTA-NOC in patients with neuroendocrine tumors: characterization of uptake in normal organs and tumor lesions. Q. J. Nucl. Med. Mol. Imaging 54, 61−67. (2) Baum, R. P., Prasad, V., Müller, D., Schuchardt, C., Orlova, A., Wennborg, A., Tolmachev, V., and Feldwisch, J. (2010) Molecular imaging of HER2-expressing malignant tumors in breast cancer patients using synthetic 111In- or 68Ga-labeled affibody molecules. J. Nucl. Med. 51, 892−897. (3) Sainz-Esteban, A., Prasad, V., and Baum, R. P. (2010) Interesting image. Pancreatic neuroendocrine tumor with involvement of the inferior mesenteric vein diagnosed by Ga-68 DOTA-TATE PET/CT. Clin. Nucl. Med. 35, 40−41. (4) Fani, M., Andre, J. P., and Maecke, H. R. (2008) 68Ga-PET: a powerful generator-based alternative to cyclotron-based PET radiopharmaceuticals. Contr. Media Mol. Imaging 3, 67−77. (5) Ballinger, J. R., and Solanki, K. K. (2011) What will be required to bring (68)Ga-labelled peptides into routine clinical use? Nucl. Med. Commun. 32, 1109−1112. (6) Tolmachev, V., Altai, M., Sandström, M., Perols, A., Eriksson Karlström, A. H., Boschetti, F., and Orlova, A. (2011) Evaluation of a maleimido derivative of NOTA for site-specific labeling of Affibody molecules. Bioconjugate Chem. 22, 894−902. (7) Fellner, M., Baum, R. P., Kubicek, V., Hermann, P., Lukes, I., Prasad, V., and Rösch, F. (2010) PET/CT imaging of osteoblastic bone metastases with 68Ga-bisphosphonates: first human study. Eur. J. Nucl. Med. Mol. Imaging 37, 834. (8) Riss, P. J., Burchardt, C., and Roesch, F. (2011) A methodical 68Ga-labelling study of DO2A-(butyl-l-tyrosine)2 with cation-exchanger post-processed 68Ga: practical aspects of radiolabelling. Contr. Media Mol. Imaging 6, 492−498. (9) Gottschaldt, M., Bohlender, C., Pospiech, A., Goerls, H., Walther, M., Mueller, D., Klette, I., Baum, R. P., and Schubert, U. S. (2009) In(III) and Ga(III) complexes of sugar substituted tripodal trisalicylidene imines: The first 68Ga labelled sugar derivative. Eur. J. Inorg. Chem., 4298−4307. (10) Meyer, G. J., Mäcke, H., Schuhmacher, J., Knapp, W. H., and Hofmann, M. (2004) 68Ga-labelled DOTA-derivatised peptide ligands. Eur. J. Nucl. Med. Mol. Imaging 31, 1097−1104. (11) Müller, D., Klette, I., Wortmann, R., and Baum, R. P. (2006) Markierung von DOTA-Peptiden mit Gallium-68 für die nuklearmedizinische Routinediagnostik. Nuklearmedizin 45, A 88−89. (12) Mueller, D., Klette, I., Gottschaldt, M., and Baum, R. P. (2009) Radiolabeling of fragile macroligands with Ga-68. J. Labeled Compd. Radiopharm. 52, S477. (13) de Blois, E., Sze Chan, H., Naidoo, C., Prince, D., Krenning, E. P., and Breeman, W. A. (2011) Characteristics of SnO2-based 68Ge/68Ga generator and aspects of radiolabelling DOTA-peptides. Appl. Radiat. Isot. 69, 308−315. (14) Zhernosekov, K. P., Filosofov, D. V., Baum, R. P., Aschoff, P., Bihl, H., Razbash, A. A., Jahn, M., Jennewein, M., and Rösch, F. (2007)

Figure 5. (A) Typical HPLC chromatogram of the final reaction mixture after 68Ga-DOTATATE synthesis using the NaCl-based labeling procedure. (B) Typical HPLC chromatogram of the final reaction mixture after 68Ga-DOTATATE synthesis using the acetone/HCl method; HPLC conditions similar to those described in Figure 2.

Table 1. Comparisons of Results from Automated Labeling Procedures Based on Cationic Exchange Cartridge Purification of 68Gaa result ± SD parameter

test method

specification

method A

method B

peptide-bound 68 Ga (%) specific activity ( MBq )

ITLC

>90%

98 ± 1

99 ± 1

dose calibrator

>20

31 ± 4

45 ± 4

acetone (ppm)

gas chromatography gas chromatography pH-meter