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

Abstract

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.

1. Introduction

Copper-64 (⁶⁴Cu) 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 ¹⁸F 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, ⁶⁴Cu also emits β⁻ and Auger electrons, enabling it to be useful for both PET imaging and radiotherapy. Moreover, the versatile coordination chemistry of ⁶⁴Cu 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 ⁶⁴Cu 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 ⁶⁴Cu as radionuclide have been applied in nuclear medicine as a means of studying their PET imaging [4,5,6]. Incorporation of ⁶⁴Cu into diacetyl-bis(N⁴-methylthiosemicarbazone) (ATSM) ligand was used for PET hypoxia imaging, such as in head and neck cancer, and cardiac conditions [7,8]. More radiotracers are in clinical development [9,10,11,12], especially those with peptides and antibodies. For example, ⁶⁴Cu-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 ⁶⁴Cu-DOTATATE provided superior image quality, and detected more lesions than ¹¹¹In-DTPA-octreotide [14]. Grubmüller, B. et al. investigated the diagnostic potential of ⁶⁴Cu-PSMA-617 in patients with prostate adenocarcinoma [15].

⁶⁴Cu has been produced at many centers [16,17,18,19]. Among the nuclear reactions examined, the ⁶⁴Ni (p, n) ⁶⁴Cu method is the best and widely used, since high production yield of the ⁶⁴Cu 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 ⁶⁴Cu on a small biomedical cyclotron using the ⁶⁴Ni (p, n) ⁶⁴Cu nuclear reaction, and ⁶⁴Cu has been produced for more than 17 years by the irradiation of electroplated enriched ⁶⁴Ni targets in this center [20,22]. The Turku PET Centre has been producing ⁶⁴Cu since 2008 using ⁶⁴Ni (p, n) ⁶⁴Cu reaction, and also handles the irradiated target, radioactive liquids and gases using automated equipment [23,24]. At the University of Wisconsin, ⁶⁴Cu and ⁶¹Co radionuclides have been simultaneously produced using the ⁶⁴Ni (p, n) ⁶⁴Cu nuclear reaction on a low energy proton-only cyclotron [25]. Ohya, T. et al. (2016) produced high-quality ⁶⁴Cu for routine use via an electrodeposited ⁶⁴Ni target, and successfully reduced the metallic impurities level of the ⁶⁴Cu product, such as Co and Ni [21]. Other nuclear reactions examined include ⁶⁴Ni (d, 2n) ⁶⁴Cu, ⁶⁴Zn (d, 2p) ⁶⁴Cu, ⁶⁴Zn (n, p) ⁶⁴Cu [26,27]. In China, researchers also have a growing interest in ⁶⁴Cu, and the demand of no-carrier added ⁶⁴Cu 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 ⁶⁴Ni solid target. The production of ⁶⁴Cu was performed on a Sumitomo HM-20 biomedical cyclotron (20 MeV) via the ⁶⁴Ni (p, n) ⁶⁴Cu reaction. The γ-ray spectroscopy of the produced ⁶⁴Cu solution was also measured to evaluate the radionuclide impurities. After ⁶⁴Cu was purified, a labeling experiment to synthesize ⁶⁴Cu-DOTA-Rituximab was performed to examine the quality of ⁶⁴Cu, 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.

2. Results and Discussion

2.1. Preparation of ⁶⁴Ni Target

In order to make ⁶⁴Cu via the nuclear reaction of ⁶⁴Ni (p, n) ⁶⁴Cu, we made the enriched ⁶⁴Ni targets on a gold (Au) disk by electrodeposition of ⁶⁴Ni from an aqueous solution of Ni(NH₃)₆²⁺ at pH = 9.05, using a robust, reliable and user-friendly apparatus (Figure 1). The Au disk (30.8 mm diameter × 1.5 mm thickness) was used as a cathode, and a platinum wire was used as an anode. A cavity of 12.1 mm diameter and 0.2 mm depth was milled into the Au disk and ⁶⁴Ni (99.32%) was plated into this cavity. The ⁶⁴Ni electroplating was performed with a constant current of 15–25 mA at 2.4–2.6 V in the aqueous solution for 48–72 h. During the electrodeposition, the green color of the ⁶⁴Ni plating solution gradually faded away and the bubbling of H₂ gas was clearly visible. When the ⁶⁴Ni plating solution became colorless, the electrodeposition was finished. The absence of Ni²⁺ was also confirmed by analytical test strips. After cleaning and drying, the plated ⁶⁴Ni on Au-disk was 70.6 ± 5.8 mg and the density of the plated ⁶⁴Ni was 61.4 ± 5.0 mg/cm², assuming a uniform thickness on the disk.

2.2. Quality Control of ⁶⁴Ni Target

The quality of the ⁶⁴Ni 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 ⁶⁴Ni layer on Au-disk. The SEM (scanning electron microscopy) image (Figure 2b) of the ⁶⁴Ni solid target showed the uniform layer of Ni on Au surface. The EDS (energy dispersive X-ray spectroscopy) (

Table 1. The metallic impurities in the plated ⁶⁴Ni target, analyzed by EDS.

Element	Wt %	At %
AsL	00.00	00.00
TcL	00.15	00.09
MnK	00.07	00.07
NiK	99.78	99.84

and Figure 2c) results showed no significant amount of metallic impurities in the ⁶⁴Ni layer. The ⁶⁴Ni target thickness on Au-disk was measured to be 10.73 μm by alpha step apparatus (Figure 2d).

2.3. Preparation of ⁶⁴Cu

After irradiation of 5 h, the ⁶⁴Ni 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 ⁶⁴Ni was washed out with 6 M HCl and collected for recycling. Due to the elevated cost of enriched ⁶⁴Ni, recycling of the target material for re-use could reduce the production cost of ⁶⁴Cu, without sacrificing the quality of subsequent ⁶⁴Cu 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 ⁶⁴Cu, which was collected and evaporated to dryness. The residue was dissolved in 0.1 M HCl for further use. The separation process of ⁶⁴Cu took about 2.5 h after irradiation.

2.4. Quality of ⁶⁴Cu

The quality of ⁶⁴Cu 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 ⁶⁴Cu solution, and showed a concentration of 6.339 ppb of Ni (0.127 μg), 4.112 ppb of Cu (0.082 μg), 5.502 ppb of Zn (0.110 μg), 0.108 ppb of Fe (0.002 μg), 0.102 ppb of Co (0.002 μg), 0.669 ppb of Ga (0.013 μg). The gamma spectrum of the produced ⁶⁴CuCl₂ solution (Figure 3) showed that the ⁶⁴Cu 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:

$$\mathrm{A}={\mathrm{A}}_0{\mathrm{e}}^{-\mathsf{\lambda }\mathrm{t}},\mathrm{lnA}=-\mathsf{\lambda }\mathrm{t}+{\mathrm{lnA}}_0,\ln \frac{\mathrm{A}}{{\mathrm{A}}_0}=-\mathsf{\lambda }\mathrm{t}$$

(1)

where A is the radioactivity of the ⁶⁴Cu at time t, A₀ is the radioactivity of the ⁶⁴Cu at 0 h, and λ represents the constant. We obtained the following equation (Figure 4):

$$\mathrm{lnA}=-0.05456\mathrm{t}+5.438$$

(2)

when $\mathrm{t}={\mathrm{T}}_{1/2}$, the $\mathrm{A}=\frac{1}{2}{\mathrm{A}}_0$

$$\ln \frac{1}{2}=-0.05456{\mathrm{T}}_{1/2}$$

(3)

The half-life of the produced ⁶⁴Cu was calculated:

$${\mathrm{T}}_{1/2}=\frac{\ln 2}{0.05456}=12.704$$

The half-life calculated (${\mathrm{T}}_{1/2}=12.704$ h) of the produced ⁶⁴Cu is in accordance with that of the radioisotope ⁶⁴Cu (${\mathrm{T}}_{1/2}=12.7$ h).

2.5. Radio-Synthesis of ⁶⁴Cu-DOTA-Rituximab and Micro-PET Imaging

To assess the quality and quantity of the produced ⁶⁴Cu, we made ⁶⁴Cu-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).

3. Materials and Methods

3.1. Materials and Reagents

High purity reagents were used for production of ⁶⁴Cu in this study. Isotopically enriched ⁶⁴Ni (99.32% ⁶⁴Ni; 0.13% ⁵⁸Ni; 0.07% ⁶⁰Ni; 0.01% ⁶¹Ni; 0.47% ⁶²Ni) was from Isoflex Company (San Francisco, CA, USA); (NH₄)₂SO₄ (99.999% metals basis) from Alfa Aesar (Ward Hill, MA, USA); concentrated HCl (99.999% trace metals basis), HNO₃ (99.999% trace metals basis), and NH₄OH (99.99% trace metals basis) were purchased from Sigma-Aldrich (St. Louis, MO, USA); AG^®1-X8 ion exchange resin was from Bio-Rad Laboratories (Hercules, CA, USA); platinum wire (99.997% metals basis) from Alfa Aesar (Ward Hill, MA, USA); Ni²⁺ analytical test strips from Qtantofix (Sigma-Aldrich, St. Louis, MO, USA); disposable PD-10 desalting columns were from GE Healthcare (Piscataway, NJ, USA).

3.2. 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 ⁶⁴Ni 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 ⁶⁴Cu was performed using a ⁶⁴Cu 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 ⁶⁴Cu 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.

3.3. Plating Solution

The ⁶⁴Ni electroplating solution was prepared as reported previously with some modifications [25]. The enriched ⁶⁴Ni 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 5 mL with 18 MΩ·cm water. The final solution was transferred to the electroplating cell for target plating.

3.4. Characterizations of Ni-64 Target

After electroplating, the ⁶⁴Ni solid target was examined by measuring ⁶⁴Ni 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 ⁶⁴Ni target was further analyzed by energy dispersive X-ray spectroscopy (EDS).

3.5. Preparation of ⁶⁴Ni Target and Irradiation

The ⁶⁴Ni targets were prepared by electrodeposition of the enriched ⁶⁴Ni (99.32%) solution prepared as described above. Electroplating of ⁶⁴Ni 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 ⁶⁴Ni 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 ⁶⁴Ni 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. ⁶⁴Cu was produced from the ⁶⁴Ni (p, n) ⁶⁴Cu nuclear reaction.

3.6. Radiochemical Separation

After irradiation, the ⁶⁴Ni 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 ⁶⁴Ni was eluted with 6 M HCl and collected for recycling, and the ⁶⁴Cu 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).

3.7. Radio-nuclide Analysis

The radionuclide identity and purity of the produced ⁶⁴CuCl₂ solution were measured using γ-ray spectroscopy (HTA Co., Ltd., Beijing, China). In addition, the identity of radioactivity of the produced ⁶⁴Cu was further confirmed by measurement of its half-life, by measuring radioactivity at different time points. A decayed sample from the produced ⁶⁴Cu was also analyzed by inductively coupled plasma-mass spectrometry (ICP-MS) for traces of metallic impurities.

3.8. Radiolabeling of DOTA-Rituximab and Micro-PET Imaging

To assess the quality of the produced ⁶⁴Cu (quantity, specific activity and purity), the synthesis of a radiotracer, ⁶⁴Cu-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 ⁶⁴CuCl₂ 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, ⁶⁴Cu-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, ⁶⁴Cu-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.

4. Conclusions

In this study, we presented the improved method for the preparation of ⁶⁴Cu, especially the improved efficiency of electroplating of ⁶⁴Ni. The ⁶⁴Ni solid target on an Au-disk has a uniform surface, with the thickness of 10.73 μm, and no metallic impurities. The total plated ⁶⁴Ni was 70.6 ± 5.8 mg and the density of the plated ⁶⁴Ni was 61.4 ± 5.0 mg/cm² (assuming a uniform thickness). After irradiation of the target and purification, the gamma spectrum of the produced ⁶⁴Cu showed its radionuclide purity to be >99%, with both peaks at 511 keV and 1346 keV. In addition, the produced ⁶⁴CuCl₂ solution has high specific activity up to 5.6 GBq/μmoL. It was further incorporated into the modified monoclonal antibody DOTA-rituximab to synthesize ⁶⁴Cu-DOTA-rituximab, which was further used successfully for micro-PET imaging.