Development of the Purification Process of Gallium-68 Eluted from Germanium-68/Gallium-68 Generator

Tossaporn Sriprapa, M.Sc.1, Thanete Doungta, MSc.2, Napamon Sritongkul, M.Sc.1, Malulee Tantawiroon, M.Sc.1

1Department of Radiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand, 2Thailand Institute of Nu clear Technology, Bangkok, Thailand.

ABSTRACT

Objective: 68Ga has a half-life of 68 minutes, with 89% of its decay is through positron emission. It is available from generator systems and possesses suitable property for labeling radioligands. These aspects make 68Ga a promising tracer for positron emission tomography (PET) imaging. This study aims to develop the purification process of the 68Ga eluates from 68Ge/68Ga generator after its recommended shelf-life and ensuring the quality through the radiolabeling process.

Materials and Methods: In this study, we explored the development of a purification method for 68Ga eluted from a 68Ge/68Ga generator before radiolabeling was investigated. Cation and anion exchange chromatography techniques were combined to remove trace amounts of competing metal ion impurities. Post-purification, the eluate’s metal contents were analyzed using inductively coupled plasma atomic emission spectroscopy (ICP-AES). Breakthrough of 68Ge was measured using a multi-channel analyzer (MCA) spectrometer with high-purity germanium (HPGe) radiation detectors. Additionally, the radiochemical purity of 68Ga-NOTA-RGD was analyzed by high-performance liquid chromatography (HPLC).

Results: Metal impurities including Fe(II), Zn(II) and Al(III) were reduced by 61%, 38% and 44% respectively. The 68Ge breakthrough was approximately ~10–3%. The labeling efficiency with NOTA-RGD, a tracer for angiogenesis imaging, resulted in an average yield of 68Ga-NOTA-RGD (not corrected for decay) of around 50%, with a radiochemical purity by HPLC of approximately 98%–99%.

Conclusion: Cation exchange in combination with anion exchange chromatography was thus proven to be an efficient method for purification of the 68Ga eluate from a 68Ge/68Ga generator prior to labeling the 68Ga PET radiotracer.

Keywords: 68Ge/68Ga generator; purification; radiolabeling; ion exchange purification; PET (Siriraj Med J 2024; 76: 90-96)

INTRODUCTION

The use of positron emission tomography (PET) has widely expanded in recent years. With its excellent resolution, high sensitivity, and potential for precise quantitative analysis, PET imaging is one of the most effective diagnostic tools in nuclear medicine today. The majority of radiopharmaceuticals used in PET are short-lived positron-emitting compounds. The four-

basic cyclotron-produced radionuclides most widely employed are: 18F 11C, 15O, and 13N. It is also possible to obtain radionuclides that emit positrons from generator systems, with some examples being 82Rb from 82Sr and 68Ga from 68Ge. While standard PET radionuclides and non-standard PET radionuclides are usually just made up of four basic radionuclides, such as 68Ga, the range

Corresponding author: Tossaporn Siriprapa E-mail: Tossaporn.sip@mahidol.ac.th

Received 1 November 2023 Revised 18 December 2023 Accepted 20 December 2023 ORCID ID:http://orcid.org/0009-0001-1626-0056 https://doi.org/10.33192/smj.v76i2.266113

All material is licensed under terms of the Creative Commons Attribution 4.0 International (CC-BY-NC-ND 4.0) license unless otherwise stated.

has expanded in recent years in both preclinical and clinical studies.1

As 68Ga decays, 89% of it is decayed through positron emission. In addition, the 68Ge parent material decays with a half-life of 270.8 days through electron capture. Currently, 68Ga is usually available from an in-house 68Ge/68Ga generator that most users will have on site but independent of an on-site cyclotron. 68Ge with a long physical half-life is ideal for clinical settings with its suitable lifespan. However, if used for more than a year, the eluting of 68Ga usually requires a hydrochloric acid solution containing 68GaCl3 as its chemical form.2,3

The use of radiolabeled peptides in nuclear oncology

is increasing. In particular, positron-emitting peptides have now been developed. 68Ga-labeled compounds are becoming increasingly popular in clinical PET.4 68Ga-labeled DOTA-peptides are the most generally used radiotracers for PET imaging, especially 68Ga- DOTA-TOC and 68Ga-DOTA-TATE in the diagnosis of neuroendocrine tumors.5,6 More recently, NOTA a commonly used bifunctional chelator, has been shown to possess a superior 68Ga-binding ability, and so 68Ga- NOTA-RGD was developed as a radiotracer for the visualization of angiogenesis.7

During the past few years, gallium has seen a change in its role in infection imaging. While 67Ga-citrate has been extensively used for the past four decades but with limitations, now 68Ga citrate and 68GaCl3 are usually used for infection imaging.8

In several 68Ge/68Ga generator systems, 68Ge is adsorbed on a wide range of solid supports, including metal oxides, such as SnO2, TiO2, and Al2O3; organic materials, e.g., pyrogallol-formaldehyde resins; and inorganic materials, e.g., silica.9-11 The main drawback of these systems is that

the 68Ga eluate is usually contaminated with long-life 68Ge and trace metallic impurities, which could potentially compete with the 68Ga ion when labeled with nanomole levels of conjugated peptides/biomolecules or other carrier ligands. In addition, the eluate from 68Ge/68Ga generators usually has a relatively large volume and high HCl concentration from 0.1–1N, which causes problems in the labeling process. Therefore, dedicated procedures to purify and concentrate 68Ga before labeling are needed.12

There has been a recent report describing a method of purifying 68Ga for performing 68Ga-labeled radiopharmaceuticals from 68Ge/68Ga generator eluates eluted with HCl/acetone mixtures on a micro-cation exchange column.13-15 In other studies, several approaches for purification and concentration have been reported. An effective post-processing technique for 68Ge/68Ga generators

using cation and anion exchange chromatography was developed to provide high 68Ga recovery, 68Ge removal, the removal of metallic impurities, lower acidity, and minimized volumes, which would all be useful for direct radiolabeling reactions with a high labeling efficiency of

68Ga-NOTA-RGD.

The aim of this study was to develop a purification method that makes use of both cation exchange and anion exchange processes to purify 68Ga eluates with a high radiochemical purity (RCP) and short purification times. The system should also provide a 68Ga eluate that can be directly used for labeling with a high radiolabeling yield and the highest radionuclidic purity.

MATERIALS AND METHODS

Purification process of the 68Ga eluate

The 68Ge/68Ga generator was purchased from iThemba LABS (Somerset West, South Africa) and had been previously checked for metallic impurities. The elution was done using sterilized 0.6N HCl12 prepared from ultra-purified hydrochloric acid and tri-distilled water. In order to elute the generator, 6 mL HCl 0.6N was used, and the eluate was divided into five equal portions of 1.2 mL each. The first portion was the non-purified eluate. The second through fifth portions were used for eluate purification by loading the eluate on the top of the cation exchange column. The column was pre-conditioned with

0.5 mL of 98% acetone/0.05 N HCl. The eluate in each column was eluted using 2 mL of 97.6% acetone with different concentrations of HCl (0.05N, 0.10N, 0.15N, and 0.2N). Each eluate from 50 mg of the cation exchange resin, which by now was in the form of [68GaCl4]-, was passed down to 50 mg of the anion exchange resin. The trapped [68GaCl4]- in the anion exchange resin was eluted with 1 mL ultra-purified water. After the purification process, the purity of the eluate in aqueous solution was investigated by inductively coupled plasma atomic emission spectrometry (ICP-AES).

Study of the metal impurities by ICP-AES

Metal impurities in the eluate were analyzed using a Spectro Arcos 165 ICP-AES system equipped with a Cetac ASX-520 autosampler. The measurements were performed using an ICP-AES spectrometer to investigate the metal ions, whereby the most prominent atomic and ionic analytical lines were chosen, including Fe at

238.104 nm, Zn at 206.2 nm, Ge at 265.118 nm, and Al at 396.153 nm. The concentrations of the following metal ions were determined by comparing them to 50, 100, 200, and 400 ppb solutions prepared from the 1000 ppb standard solution.

Germanium-68 breakthrough

Germanium-68 breakthrough was calculated by comparing the daughter radionuclide (68Ga) to the parent radionuclide (68Ge). Germanium-68 breakthrough was measured after complete 68Ga decay (<48 h) by a multi- channel analyzer (MCA) equipped with a high-purity germanium (HPGe) detector.

Radiolabeling of 68Ga-NOTA-RGD

Purified 68Ga (111 MBq in 1 mL of tri-distilled sterile water) was added to a 20 µg lyophilized NOTA-RGD kit (supplied by Jae Min Jeong Seoul National University, Jongro-gu, Seoul, Korea)16, and the pH was adjusted to

5.0 with 0.1M ammonium acetate buffer. The pH of the mixture was checked with a pH indicator strip and then the mixture was heated in a water bath at 100 oC for 15 min. After cooling, the labeled product 68Ga-NOTA- RGD was sterilized by 0.22 µm Millipore filtration.

Quality control of 68Ga-NOTA-RGD

The labeling efficiencies and radiochemical purities of the purified 68Ga-NOTA-RGD were determined by high-performance liquid chromatography (HPLC) (Agilent Tech., Series 1200) with a Phenomenex Jupiter column C-18, 5 µm, 4.6 × 250 mm. The solvents were 0.1% (m/v) trifluoroacetic acid (TFA) in deionized water (A) and 100% acetonitrile (B). Elution was carried out at a flow

rate of 1 mL/min, under UV–visible illumination (200, 280 nm) with a gamma-ray detector (Raytest Gabi Star), using the elution program in Table 1.

As a result, the peak for free 68Ga appeared at 3.5 to 4 min, whereas peaks for small particles of the radiochemical impurities appeared at 3.0 to 3.5 min, while 68Ga-NOTA- RGD showed an earlier retention time of 9.0 to 9.5 min.

RESULTS

The small amounts of Fe(III), Zn(II), Ge(IV), and Al(III) were found in the eluate. The concentration of Ge(IV) was less than 1 ppb while all the other metals were less than 1 ppm. After the purification process, Fe(II), Zn(II), and Al(III) were reduced by 61%, 38%, and 44%, respectively, compared to the initial non-purified evaluation (Table 2).

The 68Ga eluted from the 68Ge/68Ga generator usually contains small amounts of metallic impurities that represent metals that can compete with 68Ga(III) in the radiopharmaceutical labeling process, thereby adversely affecting both the 68Ga labeling yields and the specific activity of the labeled compound. These metal impurities, especially Fe(III), and the 68Ge breakthrough need to be effectively removed. Here, the concentrations of metallic ions before and after purification are shown in Table 2.

TABLE 1. HPLC gradient for the elution of 68Ga-NOTA-RGD

TABLE 2. Metal impurities concentrations in 68Ga eluates eluted with different acetone / hydrochloric acid mixtures.

68Ge breakthrough

The 68Ge breakthrough in 12 samples using a SnO2- based 68Ge/68Ga generator. After the complete decay of 68Ga (> 48 h), the quantitative measurement of 68Ge breakthrough was performed using a calibrated gamma spectrometer equipped with a coaxial (HPGe) detector.

The 68Ge breakthrough from the generator was found to be approximately 10−3% of the eluted 68Ga activity after purification. This is in accord with many publications that have reported breakthrough of the 68Ge parent radionuclide as usually less than 0.001%

Elution efficiency after purification and the labeling of RGD peptide with 68Ga

The entire purification process, including two purification steps, cation exchange and anion exchange chromatography, was completed within 30 min. The mean activity at the first elution was 2.89 ± 0.11 mCi. The radioactivity levels of 68Ga at the first elution and after passing through the cation and anion exchange columns and labeling with NOTA-RGD peptide are shown in Table 3. Without correction for the decay, the mean activities after the two purification steps and labeling were 2.00 ± 0.10, 1.74 ± 0.10, and 1.44 ± 0.10

mCi, respectively. In this study, the elution efficiencies after the purification steps were found to range between 50% to 70%. The elution efficiency decreased to about

69.02 ± 2.88% after the first purification step, 60.03 ± 2.54% after the second step, and 49.89 ± 2.49% after the labeling, see Table 3. In this study, it was approximately 70% over a processing time of not more than 30 minutes. However, the labeling efficiency was 99% and only a small amount of free 68Ga was detected when labeling NOTA-RGD peptide with the purified 68Ga, see Table 4. The complexation yield of Ga-NOTA-RGD was validated by HPLC studies. Fig 1 shows the typical HPLC pattern of 68Ga-NOTA-RGD. The 68Ga-NOTA-RGD peak was collected at a retention time of 9.0–9.5 min, while free 68Ga peak appeared at 3.5–4 min, and small

traces of radiochemical impurities at 3.0–3.5 min.

The efficacy of 68Ga for the preparation of radiopharmaceuticals for PET imaging was confirmed by radiolabeling NOTA-RGD with a very high complexation yield. The radiochemical purity (RCP) of 68Ga-NOTA- RGD was higher than 99%. The process of labeling was completed within 30 minutes. The mean labeling efficiency was 99.31 ± 0.32%, while the unlabeled 68Ga was 0.69 ± 0.32%, as shown in Table 4.

TABLE 3. Radioactivity in mCi of 68Ga at the first elution, and after the two purification steps and labeling (The percentage reduction of radioactivity is in parenthesis).

TABLE 4. Analysis of 68Ga-NOTA-RGD and unlabeled 68Ga by HPLC.

Fig 1. HPLC chromatogram patterns of 68Ga-NOTA-RGD.

CONCLUSION AND DISCUSSION

The 68Ge/68Ga generator used in this study was a SnO2-based generator. By measuring the metallic impurities by ICP-AES, it was verified that metallic impurities were present that could interfere with the formation of Ga(III) complexes. Therefore, before the radiolabeling of peptides, the 68Ga eluate would need to be purified using a cationic exchange column, an anionic exchange column, or both. In this study, 68Ga solutions were purified on both cation and anion exchange columns. We eluted the 68Ga eluate using different concentrations of acetone/ hydrochloric acid mixtures after each transfer. Schultz et al.17 used different sets of chromatographic columns, 50W X8 cation, and UTEVA resin, and eluted with 0.1M HCl. Metal analysis by ICP-AES demonstrated that the stable metals were reduced to less than 0.2 ppm, but Fe(III) could not be removed, while the breakthrough of 68Ge was less than 0.02% of the 68Ga activity.

Germanium-68 is strongly absorbed by metal oxides or organic materials, making 68Ge breakthrough highly unlikely. However, the metal impurities from 68Ge breakthrough in the eluate are lesser problems compared to patient exposure to radiation when used for the radiolabeling of peptides or other biomolecules. As a minimum, the radionuclidic purity of 68Ga chloride solution should be limited to 99.9% of the total radioactivity, whereas 68Ge should not exceed 0.001% (EU Pharm). Some have even reported values less than 10–4% to 10–5%. Konstantin et al.18 reported that the initial amount of 68Ge(IV) was decreased by a factor of 104 when using a TiO2-based generator. Roesch20 reported that 68Ge breakthrough levels ranged from 0.01% to 0.001% for fresh generators, but they increase with extended use. Since the 68Ge breakthrough has been shown to increase over the lifetime of the generator and our generator had been used for more than 18 months, our presented result of 10–3% of the eluted 68Ga activity is considered an acceptable result. This means our generator also fulfills the requirement of the European Pharmacopeia (EU) concerning the radionuclide purity for 68Ga of 99.9%. Concerning the radiation absorbed dose, a recent publication reported that 68Ge is rapidly excreted in the urine, which greatly diminishes the potential radiation absorbed dose. Lin M et al.21 demonstrated that the elution of 68Ge from a commercial titanium-dioxide-based 68Ge/68Ga generator resulted in markedly low 68Ge breakthrough, in the order of 14 to 25 nCi. When labeled with DOTATOC, spectroscopic analysis of the synthesis components demonstrated that the 68Ge breakthrough in the final products was quantitatively removed. Sudbrock et al.22 reported that the content of long-lived 68Ge breakthrough increased

to more than 100 ppm over the entire period of use of the generator, while the chelator DOTA eliminated 68Ge efficiently during labeling. The maximum 68Ge activity found in the labeled product (below 10 Bq) and the effective doses received by the patient from 68Ge in the 68Ga-DOTATATE final product were lower than 0.1 μSv, meaning practically insignificant for patients.

After the elution of 68Ga, the activity of 68Ga was measured immediately using a calibrated ionization chamber to determine the elution efficiency. The 68Ga elution yields dropped with increasing its usage frequency or shelf-life. In this study, the elution yield of our generator was found to be lower than 50% because it has been used for almost 18 months. Roesch23 reported that the 68Ga-eluted yields range from about 70% to 80% for fresh generators, but these decrease over time. The initial yield of the generator has been reported to range from 75% to 100% and the long-term yield from 60% to more than 80%. Patrascu et al.24 reported an elution efficiency of 80% and Konstantin et al.18 reported an initial activity of more than 97% from a TiO2-based generator.

The time spent processing the generator eluate, synthesizing the labeled product, and purifying it reduced the production yield. In this study without correction for the decay, the final yield of 68Ga-NOTA-RGD peptide was less than 50% (49.89 ± 2.49%). A final yield of 46 ± 5% for the 68Ga-labeled DOTA-conjugated octreotide was reported by Roesch and Filosofov.25 Further, the decay-corrected yields of 68Ga radiopharmaceuticals did not exceed 60% to 70%.

For the clinical application of 68Ga produced by 68Ge/68Ga generators, it is important to obtain 68Ga in a purified chemical form, maximize the elution yield of 68Ga, and reduce the elution volume while maintaining a permissible level of 68Ge impurity in the eluate. There is a possibility of regularly eluting 68Ge/68Ga from the generator in a way that provides an acceptable radioactive concentration, yield, and purity. It was reported by Asti et al.19 that the concentration of 68Ge breakthrough increased with time, with approximately a 15% increase per month, ranging from 1.1×10−2% to 2.6×10−2% of the 68Ga activity within their 7 months of evaluation. Moreover, the elution yields of 68Ga from these generators decreased from 82% to 69% when elution was repeated, i.e., 100 times, over the period of 7 months.19

The RCP of the 68Ga-labeled product should be greater than 99%, which would result in a high efficiency of radiolabeling with a small volume and low acidity. In the present study, the efficacy of 68Ga for the preparation of radiopharmaceuticals for PET imaging was confirmed by radiolabeling NOTA-RGD with a very high yield.

At present, an automatic synthesis module to produce 68Ga-NOTA-RGD may be used. The module was connected to a line elution 68Ge/68Ga generator with a purification part and controlled by a personal computer program for easy production and to make it suitable for routine work. However, a variety of post-processing methods, such as anionic exchange and cationic exchange purification, are required to purify the eluate. It is also of particular importance for the labeled products to have high specific activities.

ACKNOWLEDGEMENTS

The dissertation was financially supported under Research Fellowship, provided by Siriraj Graduate Scholarship. We are grateful for Radioisotope Center, Thailand Institute of Nuclear Technology for the permission to include copyrighted photographs as part of my thesis. I would like to thank Mr. Jatupol Sangsuriyan and Miss Nipavan Poramatikul for their valuable and constructive suggestions during the planning of this research work.

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