Establishing Reliable Cu-64 Production Process: From Target Plating to Molecular Specific Tumor Micro-PET Imaging

Copper-64 is a useful radioisotope for positron emission tomography (PET). Due to the wide range of applications, the demand of 64Cu with low metallic impurities is increasing. Here we report a simple method for the efficient production of high specific activity 64Cu using a cyclotron for biomedical application. We designed new equipment based on the plating of enriched 64Ni as the target, and used automated ion exchange chromatography to purify copper-64 efficiently after irradiation and dissolution of the target in good radiochemical and chemical yield and purity. The 64Cu radionuclide produced using 99.32% enriched 64Ni with a density of 61.4 ± 5.0 mg/cm2, reaching a total radioactivity greater than 200 mCi, with specific activity up to 5.6 GBq/μmoL. It was further incorporated into modified monoclonal antibody DOTA-rituximab to synthesize 64Cu-DOTA-rituximab, which was used successfully for micro-PET imaging.


Introduction
Copper-64 ( 64 Cu) is an attractive radionuclide of considerable interest for positron emission tomography (PET) imaging and radiotherapy due to its intrinsic physical and chemical properties. It has high spatial resolution comparable to 18 F radionuclide, with comparable average free travel distance for their generated positrons (R ave. (β + ) = 0.70 and 0.69 mm, respectively), due to their comparable positron energy (0.656 MeV and 0.635 MeV, respectively) [1,2]. It has a relatively long half-life of 12.7 h, compared with fluorine-18 (t 1/2 = 110 min) and carbon-11 (t 1/2 = 20.4 min). In addition, 64 Cu also emits β − and Auger electrons, enabling it to be useful for both PET imaging and radiotherapy. Moreover, the versatile coordination chemistry of 64 Cu allows for its reaction with a wide variety of chelator systems, such as DOTA, NOTA, TETA and CB-TE2A, that can be linked to antibodies, peptides and nanoparticles [3]. The 64 Cu radioisotope can be used for the design and synthesis of a wide range of radio-probes, providing attractive candidates for PET imaging.
A number of radiotracers involving 64 Cu as radionuclide have been applied in nuclear medicine as a means of studying their PET imaging [4][5][6]. Incorporation of 64 Cu into diacetyl-bis(N 4 -methylthiosemicarbazone) (ATSM) ligand was used for PET hypoxia imaging, such as in head and neck cancer, and cardiac conditions [7,8].
For example, 64 Cu-DOTA-trastuzumab was used to conduct PET imaging of HER2-positive lesions in patients with primary and metastatic breast cancer [13]. The PET image of 64 Cu-DOTATATE provided superior image quality, and detected more lesions than 111 In-DTPA-octreotide [14]. Grubmüller, B. et al. investigated the diagnostic potential of 64 Cu-PSMA-617 in patients with prostate adenocarcinoma [15]. 64 Cu has been produced at many centers [16][17][18][19]. Among the nuclear reactions examined, the 64 Ni (p, n) 64 Cu method is the best and widely used, since high production yield of the 64 Cu can be obtained with low energy protons in this route [20,21]. At Washington University, an effective method was investigated to produce high specific activity 64 Cu on a small biomedical cyclotron using the 64 Ni (p, n) 64 Cu nuclear reaction, and 64 Cu has been produced for more than 17 years by the irradiation of electroplated enriched 64 Ni targets in this center [20,22]. The Turku PET Centre has been producing 64 Cu since 2008 using 64 Ni (p, n) 64 Cu reaction, and also handles the irradiated target, radioactive liquids and gases using automated equipment [23,24]. At the University of Wisconsin, 64 Cu and 61 Co radionuclides have been simultaneously produced using the 64 Ni (p, n) 64 Cu nuclear reaction on a low energy proton-only cyclotron [25]. Ohya, T. et al. (2016) produced high-quality 64 Cu for routine use via an electrodeposited 64 Ni target, and successfully reduced the metallic impurities level of the 64 Cu product, such as Co and Ni [21]. Other nuclear reactions examined include 64 Ni (d, 2n) 64 Cu, 64 Zn (d, 2p) 64 Cu, 64 Zn (n, p) 64 Cu [26,27]. In China, researchers also have a growing interest in 64 Cu, and the demand of no-carrier added 64 Cu has started to increase.
Here we report a robust, reliable and user-friendly plating vessel, which can be used for the effective preparation of the 64 Ni solid target. The production of 64 Cu was performed on a Sumitomo HM-20 biomedical cyclotron (20 MeV) via the 64 Ni (p, n) 64 Cu reaction. The γ-ray spectroscopy of the produced 64 Cu solution was also measured to evaluate the radionuclide impurities. After 64 Cu was purified, a labeling experiment to synthesize 64 Cu-DOTA-Rituximab was performed to examine the quality of 64 Cu, including labeling yield and radiochemical purity of the radiotracer, which targets the CD20 antigen, which is expressed on B cell lymphocytes and in the majority of non-Hodgkin's lymphoma (NHL) [28]. The synthesized radiotracer was further examined by micro-PET imaging using SCID (severe combined immune deficiency) mice bearing Ramos RA1 tumors which overexpress CD20 antigen.

Preparation of 64 Ni Target
In order to make 64 Cu via the nuclear reaction of 64 Ni (p, n) 64 Cu, we made the enriched 64 Ni targets on a gold (Au) disk by electrodeposition of 64 Ni from an aqueous solution of Ni(NH 3 ) 6 2+ at pH = 9.05, using a robust, reliable and user-friendly apparatus ( Figure 1).

Quality Control of 64 Ni Target
The quality of the 64 Ni solid target prepared above was evaluated by a number of physical techniques, to determine the uniformity, to characterize the metallic impurities, and to measure the thickness of 64 Ni layer on Au-disk. The SEM (scanning electron microscopy) image (Figure 2b) of the 64 Ni solid target showed the uniform layer of Ni on Au surface. The EDS (energy dispersive X-ray spectroscopy) ( Table 1 and Figure 2c) results showed no significant amount of metallic impurities in the 64 Ni layer. The 64 Ni target thickness on Au-disk was measured to be 10.73 μm by alpha step apparatus ( Figure 2d).

Quality Control of 64 Ni Target
The quality of the 64 Ni solid target prepared above was evaluated by a number of physical techniques, to determine the uniformity, to characterize the metallic impurities, and to measure the thickness of 64 Ni layer on Au-disk. The SEM (scanning electron microscopy) image (Figure 2b) of the 64 Ni solid target showed the uniform layer of Ni on Au surface. The EDS (energy dispersive X-ray spectroscopy) ( Table 1 and Figure 2c) results showed no significant amount of metallic impurities in the 64 Ni layer. The 64 Ni target thickness on Au-disk was measured to be 10.73 µm by alpha step apparatus ( Figure 2d).

Quality Control of 64 Ni Target
The quality of the 64 Ni solid target prepared above was evaluated by a number of physical techniques, to determine the uniformity, to characterize the metallic impurities, and to measure the thickness of 64 Ni layer on Au-disk. The SEM (scanning electron microscopy) image ( Figure 2b) of the 64 Ni solid target showed the uniform layer of Ni on Au surface. The EDS (energy dispersive X-ray spectroscopy) ( Table 1 and Figure 2c) results showed no significant amount of metallic impurities in the 64 Ni layer. The 64 Ni target thickness on Au-disk was measured to be 10.73 μm by alpha step apparatus ( Figure 2d).

Preparation of 64 Cu
After irradiation of 5 h, the 64 Ni target was dissolved in 6 M hydrochloride acid, and then the solution was load to an anion exchange column to separate into different components. The 64 Ni was washed out with 6 M HCl and collected for recycling. Due to the elevated cost of enriched 64 Ni, recycling of the target material for re-use could reduce the production cost of 64 Cu, without sacrificing the quality of subsequent 64 Cu production. When the eluted was switched to 1 M HCl, the first band coming out was co-produced cobalt radioisotopes (approximately 1 mL), and the second was the 64 Cu, which was collected and evaporated to dryness. The residue was dissolved in 0.1 M HCl for further use. The separation process of 64 Cu took about 2.5 h after irradiation.

Quality of 64 Cu
The quality of 64 Cu produced was evaluated by analysis of its metallic impurities and measurement of half-life of its radioactivity. The inductively coupled plasma-mass spectrometry (ICP-MS) analysis was used to evaluate the amount of metallic impurities in a decayed 64 (Figure 3) showed that the 64 Cu radionuclide purity was >99%. The half-life of the produced radioactivity was determined by the radioactivity measured at different time points into the following equations: where A is the radioactivity of the 64 Cu at time t, A 0 is the radioactivity of the 64 Cu at 0 h, and λ represents the constant. We obtained the following equation (Figure 4): The half-life of the produced 64 Cu was calculated: The half-life calculated (T 1/2 = 12.704 h) of the produced 64 Cu is in accordance with that of the radioisotope 64 Cu (T 1/2 = 12.7 h).

Preparation of 64 Cu
After irradiation of 5 h, the 64 Ni target was dissolved in 6 M hydrochloride acid, and then the solution was load to an anion exchange column to separate into different components. The 64 Ni was washed out with 6 M HCl and collected for recycling. Due to the elevated cost of enriched 64 Ni, recycling of the target material for re-use could reduce the production cost of 64 Cu, without sacrificing the quality of subsequent 64 Cu production. When the eluted was switched to 1 M HCl, the first band coming out was co-produced cobalt radioisotopes (approximately 1 mL), and the second was the 64 Cu, which was collected and evaporated to dryness. The residue was dissolved in 0.1 M HCl for further use. The separation process of 64 Cu took about 2.5 h after irradiation.

Quality of 64 Cu
The quality of 64 Cu produced was evaluated by analysis of its metallic impurities and measurement of half-life of its radioactivity. The inductively coupled plasma-mass spectrometry (ICP-MS) analysis was used to evaluate the amount of metallic impurities in a decayed 64 where A is the radioactivity of the 64 Cu at time t, A0 is the radioactivity of the 64 Cu at 0 h, and λ represents the constant. We obtained the following equation (Figure 4): when t T / , the A A ln The half-life of the produced 64 Cu was calculated: The half-life calculated (T / 12.704 h) of the produced 64 Cu is in accordance with that of the radioisotope 64 Cu (T / 12.7 h).

Radio-Synthesis of 64 Cu-DOTA-Rituximab and Micro-PET Imaging
To assess the quality and quantity of the produced 64 Cu, we made 64 Cu-DOTA-rituximab ( Figure 5), with high chemical yield, high radiochemical purity, and high specific activity. Micro-PET imaging of the radiotracer in mice bearing Ramos RA1 tumors clearly showed the tumor at 24 h and 60 h post-injection, with excellent resolution and clarity at the latter ( Figure 6).

Radio-Synthesis of 64 Cu-DOTA-Rituximab and Micro-PET Imaging
To assess the quality and quantity of the produced 64 Cu, we made 64 Cu-DOTA-rituximab ( Figure 5), with high chemical yield, high radiochemical purity, and high specific activity. Micro-PET imaging of the radiotracer in mice bearing Ramos RA1 tumors clearly showed the tumor at 24 h and 60 h post-injection, with excellent resolution and clarity at the latter ( Figure 6).

Radio-Synthesis of 64 Cu-DOTA-Rituximab and Micro-PET Imaging
To assess the quality and quantity of the produced 64 Cu, we made 64 Cu-DOTA-rituximab ( Figure 5), with high chemical yield, high radiochemical purity, and high specific activity. Micro-PET imaging of the radiotracer in mice bearing Ramos RA1 tumors clearly showed the tumor at 24 h and 60 h post-injection, with excellent resolution and clarity at the latter ( Figure 6).

Materials and Reagents
High purity reagents were used for production of 64

Equipment
The alpha step apparatus (Alpha-step IQ, KLA-Tencor, Milpitas, CA, USA), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS) (1910FE, AMRAY, Pawtucket, RI, USA) were used to characterize the quality of the 64 Ni solid target produced in this study. The irradiation experiments were performed using a Sumitomo HM-20 cyclotron (20 MeV, Sumitomo Heavy Industries, Ltd., Tokyo, Japan). The separation of 64 Cu was performed using a 64 Cu separation system (Industrial Equipment Division, Sumitomo Heavy Industries, Ltd., Tokyo, Japan). Inductively coupled plasma-mass spectrometry (ICP-MS) (ELEMENT XR mass spectrometer, Thermo Fisher, Bremen, Germany) was used to analyze the purity of the 64 Cu sample. The Agilent Technologies 1200 series of high performance liquid chromatography (HPLC) system (Agilent, Lake Forest, CA, USA) equipped with both a UV absorption detector and a B-Fc-1000 HPLC radioactivity detector (Bioscan, Washington, DC, USA) and the radioactive thin-layer chromatography scanner (Radio-TLC) (Bioscan, IAR-2000, Washington, DC, USA) were used to analyze the radiochemical purity of tracers.

Plating Solution
The 64 Ni electroplating solution was prepared as reported previously with some modifications [25]. The enriched 64 Ni metal (65-80 mg) was dissolved in 5 mL of warm 6 M HNO3. After the metal was completely dissolved, the solution was then evaporated to dryness under vacuum. The green-colored residue was dissolved in 300 μL of concentrated H2SO4 and the solution then diluted with 2 mL of 18 MΩ·cm water (Milli-Q Waters, Millipore Corporation, Billerica, MA, USA) slowly and carefully. The pH of the solution was adjusted to 9.05 ± 0.05 by adding about 1.5 mL concentrated NH4OH. To this solution, ~300 mg of (NH4)2SO4 was added and the volume of the solution was adjusted to

Materials and Reagents
High purity reagents were used for production of 64

Equipment
The alpha step apparatus (Alpha-step IQ, KLA-Tencor, Milpitas, CA, USA), scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS) (1910FE, AMRAY, Pawtucket, RI, USA) were used to characterize the quality of the 64 Ni solid target produced in this study. The irradiation experiments were performed using a Sumitomo HM-20 cyclotron (20 MeV, Sumitomo Heavy Industries, Ltd., Tokyo, Japan). The separation of 64 Cu was performed using a 64 Cu separation system (Industrial Equipment Division, Sumitomo Heavy Industries, Ltd., Tokyo, Japan). Inductively coupled plasma-mass spectrometry (ICP-MS) (ELEMENT XR mass spectrometer, Thermo Fisher, Bremen, Germany) was used to analyze the purity of the 64 Cu sample. The Agilent Technologies 1200 series of high performance liquid chromatography (HPLC) system (Agilent, Lake Forest, CA, USA) equipped with both a UV absorption detector and a B-Fc-1000 HPLC radioactivity detector (Bioscan, Washington, DC, USA) and the radioactive thin-layer chromatography scanner (Radio-TLC) (Bioscan, IAR-2000, Washington, DC, USA) were used to analyze the radiochemical purity of tracers.

Plating Solution
The 64 Ni electroplating solution was prepared as reported previously with some modifications [25]. The enriched 64 Ni metal (65-80 mg) was dissolved in 5 mL of warm 6 M HNO 3 . After the metal was completely dissolved, the solution was then evaporated to dryness under vacuum. The green-colored residue was dissolved in 300 µL of concentrated H 2 SO 4 and the solution then diluted with 2 mL of 18 MΩ·cm water (Milli-Q Waters, Millipore Corporation, Billerica, MA, USA) slowly and carefully. The pH of the solution was adjusted to 9.05 ± 0.05 by adding about 1.5 mL concentrated NH 4 OH.
To this solution,~300 mg of (NH4) 2 SO 4 was added and the volume of the solution was adjusted to 5 mL with 18 MΩ·cm water. The final solution was transferred to the electroplating cell for target plating.

Characterizations of Ni-64 Target
After electroplating, the 64 Ni solid target was examined by measuring 64 Ni thickness, composition and structure. The thickness was measured by alpha step apparatus, the composition was measured by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), and the structure was characterized by scanning electron microscopy (SEM). Meanwhile, the identity of metallic impurities of the 64 Ni target was further analyzed by energy dispersive X-ray spectroscopy (EDS).

Preparation of 64 Ni Target and Irradiation
The 64 Ni targets were prepared by electrodeposition of the enriched 64 Ni (99.32%) solution prepared as described above. Electroplating of 64 Ni was realized using a plating vessel of our own design and manufacture (Figure 1), where the Au disk was used as a cathode and platinum wire as an anode. The electrodeposition of 64 Ni was achieved at 2.4-2.6 V and 15-25 mA with the platinum anode at~1 cm from the Au disk electrode. This process took 48-72 h. The enriched 64 Ni target was irradiated at 12.5 MeV (which decreased by Al) with 20 µA current on a Sumitomo HM-20 cyclotron (20 MeV) for about 5-7 h. 64 Cu was produced from the 64 Ni (p, n) 64 Cu nuclear reaction.

Radiochemical Separation
After irradiation, the 64 Ni target was placed into the dissolving bath of 6 M HCl. The complete dissolution of the target material took about 40 min under heating, and then the solution was loaded on an anion exchange column (AG 1-X8) pretreated with 18 MΩ·cm water and 6 M HCl, sequentially. The enriched 64 Ni was eluted with 6 M HCl and collected for recycling, and the 64 Cu fraction was eluted with 1 M HCl, which was further evaporated to dryness. The residue was finally dissolved in 0.1 M HCl for further use. All of these procedures were performed using an automated system and carried out in a hot cell with remote control (Figure 7, Sumitomo Heavy Industries, Ltd., Tokyo, Japan).

Characterizations of Ni-64 Target
After electroplating, the 64 Ni solid target was examined by measuring 64 Ni thickness, composition and structure. The thickness was measured by alpha step apparatus, the composition was measured by scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), and the structure was characterized by scanning electron microscopy (SEM). Meanwhile, the identity of metallic impurities of the 64 Ni target was further analyzed by energy dispersive X-ray spectroscopy (EDS).

Preparation of 64 Ni Target and Irradiation
The 64 Ni targets were prepared by electrodeposition of the enriched 64 Ni (99.32%) solution prepared as described above. Electroplating of 64 Ni was realized using a plating vessel of our own design and manufacture (Figure 1), where the Au disk was used as a cathode and platinum wire as an anode. The electrodeposition of 64 Ni was achieved at 2.4-2.6 V and 15-25 mA with the platinum anode at ~1 cm from the Au disk electrode. This process took 48-72 h. The enriched 64 Ni target was irradiated at 12.5 MeV (which decreased by Al) with 20 μA current on a Sumitomo HM-20 cyclotron (20 MeV) for about 5-7 h. 64 Cu was produced from the 64 Ni (p, n) 64 Cu nuclear reaction.

Radiochemical Separation
After irradiation, the 64 Ni target was placed into the dissolving bath of 6 M HCl. The complete dissolution of the target material took about 40 min under heating, and then the solution was loaded on an anion exchange column (AG 1-X8) pretreated with 18 MΩ·cm water and 6 M HCl, sequentially. The enriched 64 Ni was eluted with 6 M HCl and collected for recycling, and the 64 Cu fraction was eluted with 1 M HCl, which was further evaporated to dryness. The residue was finally dissolved in 0.1 M HCl for further use. All of these procedures were performed using an automated system and carried out in a hot cell with remote control (Figure 7, Sumitomo Heavy Industries, Ltd., Tokyo, Japan).

Radio-nuclide Analysis
The radionuclide identity and purity of the produced 64 CuCl 2 solution were measured using γ-ray spectroscopy (HTA Co., Ltd., Beijing, China). In addition, the identity of radioactivity of the produced 64 Cu was further confirmed by measurement of its half-life, by measuring radioactivity at different time points. A decayed sample from the produced 64 Cu was also analyzed by inductively coupled plasma-mass spectrometry (ICP-MS) for traces of metallic impurities.

Radiolabeling of DOTA-Rituximab and Micro-PET Imaging
To assess the quality of the produced 64 Cu (quantity, specific activity and purity), the synthesis of a radiotracer, 64 Cu-DOTA-rituximab, was performed. Here, the site-specific modification of monoclonal antibody DOTA-rituximab which contains two DOTA chelator in each antibody was used as we previously reported [29]. The 64 CuCl 2 solution prepared above was reacted with DOTA-rituximab in a solution of pH = 5.5 at RT for 30 min. After incubation, the radiotracer was purified using a PD-10 desalting column, and characterized by Radio-TLC and Radio-HPLC, to determine the labeling yield and radiochemical purity. The identity of the radiotracer, 64 Cu-DOTA-rituximab, was further evaluated by PET imaging, which was carried out on a micro-PET rodent model scanner as reported earlier [29]. After formulation, 64 Cu-DOTA-rituximab (0.5 mCi) was injected intravenously into the tail vein of the mice bearing Ramos RA1 tumors (n = 3), which overexpress CD20 antigen, and the animals were imaged with micro-PET at both 24 and 60 h post-injection.

Conclusions
In this study, we presented the improved method for the preparation of 64 Cu, especially the improved efficiency of electroplating of 64 Ni. The 64 Ni solid target on an Au-disk has a uniform surface, with the thickness of 10.73 µm, and no metallic impurities. The total plated 64 Ni was 70.6 ± 5.8 mg and the density of the plated 64 Ni was 61.4 ± 5.0 mg/cm 2 (assuming a uniform thickness). After irradiation of the target and purification, the gamma spectrum of the produced 64 Cu showed its radionuclide purity to be >99%, with both peaks at 511 keV and 1346 keV. In addition, the produced 64 CuCl 2 solution has high specific activity up to 5.6 GBq/µmoL. It was further incorporated into the modified monoclonal antibody DOTA-rituximab to synthesize 64 Cu-DOTA-rituximab, which was further used successfully for micro-PET imaging.