An Experimental Generator for Production of High-Purity 212Pb for Use in Radiopharmaceuticals

Visual Abstract

Lead-212 ( 212 Pb; half-life, 10.6 h), a b-emitter itself, is an in vivo generator of a-particles through the a-emitting progenies 212 Bi and 212 Po. Convenient chelation chemistry makes 212 Pb suitable for targeted a-therapy (1). However, the radiolabeling of targeting agents should preferably be performed on-site because of the short half-life of 212 Pb. Rapid and efficient processes are required to ensure sufficient 212 Pb availability for end users.
As a member of the thorium series ( Fig. 1), 212 Pb can be obtained from generators that contain the longer-lived mother nuclides 228 Th (half-life, 1.9 y) or 224 Ra (half-life, 3.6 d). Current generators are based on isolating 212 Pb from 224 Ra or 228 Th through several purification steps. 224 Ra has become the preferred radionuclide source over 228 Th to minimize radiation hazards (1). A generator used to supply 212 Pb for clinical trials by Orano Med is based on 224 Ra immobilized on a cation-exchange column from which 212 Pb can be eluted (2)(3)(4). The eluate is then evaporated and treated several times with concentrated acid before the final solution is ready for radiolabeling (3). A similar generator purchased from Oak Ridge National Laboratory was integrated into an automated synthesis module, where 212 Pb was eluted in dilute HCl for labeling of peptides (5). An alternative method that avoids purification of 212 Pb from the generator source material uses a solution of 224 Ra/ 212 Pb in equilibrium directly for the radiolabeling process (6,7). However, this procedure still requires a final purification step to remove 224 Ra and unconjugated daughters.
A second approach is based on radon emanation, which involves obtaining 212 Pb from gaseous 220 Rn (half-life, 55.6 s) emanated from the decaying ( 228 Th/) 224 Ra parent. Thus, 212 Pb can be isolated from parent nuclides without the need for dedicated equipment for the separation process. Hassfjell and Hoff reported a generator comprising a 228 Th source distributed within barium stearate and stored in a housing chamber connected to a vacuum pump (8). The source could be slid into the collection chamber via a gate valve. The generator experienced a relatively poor yield (11%-50%) because of radiation damage of the source when 40-50 MBq of 228 Th were used. Other examples are based on 2-compartment systems in which 220 Rn is transferred from a source chamber with parent nuclides into a collector chamber by airflow (9)(10)(11). These generators require significant effort and advanced equipment and have been tested in only small-scale production (#2 MBq). Another drawback of such 2-compartment systems is that 220 Rn may decay before reaching the collector chamber, potentially resulting in low 212 Pb yields (9).
Here, we report a novel single-chamber generator based on 220 Rn emanation from decaying 224 Ra or 228 Th to produce high yields of 212 Pb for radiolabeling of ligands and monoclonal antibodies (mAbs). The generator is compact and user-friendly-key considerations for a shippable device that can be operated by the staff at a nuclear medicine facility.

MATERIALS AND METHODS
The 228 Th/ 224 Ra/ 212 Pb Generator An earlier generation of the generator was previously reported (12), but the recent version was optimized to increase output capacity and reduce the risk of cross-contamination. The generator, consisting of a 100-mL glass flask standing upside down, with the radionuclide source contained in the screw cap ( Fig. 2) (13), was kept at room temperature the entire time. 228 Th (Eckert and Ziegler or Oak Ridge National Laboratory) or 224 Ra (prepared as previously described (7)) in 100-200 mL of 0.1-1 M HCl was applied to approximately 0.2 g of porous quartz wool (ProQuarz GmbH). The quartz wool was placed on a small plastic cap covered in aluminum foil to minimize 220 Rn retention and secured inside the screw cap (Fig. 2). During 228 Th/ 224 Ra decay, the short-lived 220 Rn emanated from the quartz wool, followed by adsorption of the longer-lived 212 Pb daughter onto the interior surfaces of the flask. After approximately 2 d, the flask was carefully replaced with a clean flask to harvest 212 Pb and reuse the generator, ensuring no cross-contamination from the source. To extract the 212 Pb, 0.5-1 mL of 0.1 M HCl solution was added, and the flask was carefully swirled to cover the inner surface for about 5 min before the solution was collected.

Radioactivity Measurements
A pure source of 224 Ra reaches transient equilibrium with 212 Pb after 2 d. We evaluated the yield of the 224 Ra-based generator when 212 Pb was harvested after 2-3 d, or as the average yield for the 228 Th-based generator when 1 generator was used multiple times with at least a 2-d interval. The yield was defined as the percentage of 212 Pb activity adsorbed to the flask relative to parent 224 Ra or 228 Th. The yield was also evaluated for generators that were milked for the second time. Radioactivity was quantified by a radioisotope dose calibrator (CRC-25R; Capintec Inc.) (12).
The breakthrough of 224 Ra or 228 Th in the washout solution at harvesting was quantified indirectly through the 212 Pb activity of decayed samples-activity that was measured in the 60-to 110-keV window on a g-counter (automatic g-counter; Hidex Oy) (12). The details of the measurements and calculations are described in Supplemental Section 1 (supplemental materials are available at http://jnm.snmjournals.org). 220 Rn emanation from the generator and the dose rate resulting from x-rays and g-rays were evaluated for radiation safety purposes as described in detail in Supplemental Section 2.

Generator Yield, Performance, and Feasibility
The single-chamber 212 Pb generator was easy to use and handle. The 212 Pb solution could be extracted at regular intervals, and the generator cap could be transferred to a clean flask each time for reuse. Its small size allowed measurement in a standard ionization chamber dose calibrator. from the 228 Th-based generator, the measured radioactivity was below the quantification limit of the instrument.

Radiation Safety Aspects
Our evaluation of generator integrity did not indicate any escape of 220 Rn when the generator was closed. However, radon exposure from the generator is a potential radiation safety concern when the generator is opened, because the half-life of 220 Rn is long enough for the gas to reach its surroundings. In the experimental setup in which a 1-MBq 224 Ra-based generator was opened inside a sealed bag for 10 s, approximately 11% of the available 220 Rn escaped (Supplemental Section 2).
Exposure to x-rays and g-rays is another potential safety concern. The measurements on the surface of a 2-cm lead shield showed an average dose rate of 20 mSv/h per MBq  of 228 Th. The dose rate was considerably reduced to 2.3 mSv/h per MBq for a 5-cm lead shield and to 0.7 mSv/h per MBq for a 7-cm lead shield.

Radiochemical Purity of Radioconjugates
The 212 Pb extracted from 224 Ra-based generators was used to radiolabel TCMC-conjugated ligands and mAbs with a high and reproducible radiochemical purity for all tested compounds ( Table 2).

DISCUSSION
Here, we present an experimental 212 Pb generator that is compact, easy to use, and operable without advanced equipment or hazardous chemicals. These considerations are important for the convenient and efficient routine production of 212 Pb in clinical applications.
To our knowledge, there are no existing 212 Pb generators that meet these criteria entirely (1,14,15). The 228 Th-based generator bypasses the 224 Ra separation step from 228 Th while being a longer-lived device that facilitates upscaled production of 212 Pb at an industrial scale. Results show that a single 228 Th-based generator could be milked every 2-5 d to routinely supply high-purity 212 Pb for research and development. Radiopharmacies and hospitals must consider the exemption limit of 228 Th-which is a tenth of that of 224 Ra and 212 Pb in the European Union and United States-when applying for permits for certified use. No well-defined criteria for an acceptable level of 228 Th impurity in a radiopharmaceutical exist, but for 7 of 8 samples, the values were below the acceptance limit (,0.002%) for the impurity level of another therapeutic radiopharmaceutical that is described in the European Pharmacopoeia (16). Assuming a 100-MBq patient dose, the value is comparable to the effective dose-derived annual limit of intake of 228 Th (17). The breakthrough of 224 Ra from the 224 Ra-based generator was comparable to the current state of the art (14). Hence, a clinically relevant purity is achievable with the presented technology.
Decay of 228 Th/ 224 Ra results in an increasing accumulation of the stable daughter nuclide 208 Pb in the generator, which potentially competes with 212 Pb in radiolabeling procedures. The cumulative amount of 208 Pb in the extracted 212 Pb can be estimated on the basis of the generator yield ( Fig. 3; Supplemental Section 4). In terms of mAb binding, these fractions may not influence radiochemical purity, as only 1 in about 2,000 mAbs needs to be bound by a 212 Pb atom for a clinically relevant specific activity (18).
The current generator is a prototype from which a limited number of 212 Pb extractions have been performed. Along with upscaling, the radiation safety and yield may be areas for improvement in future studies. Both issues can be addressed by design considerations. The source-holding material, its size and volume, and the inner surface area of the generator can be optimized to increase the levels of 212 Pb depositing onto the surface. It should be verified that the holding material is not affected when one is working with higher radioactivity levels. A closed system with an integrated shielding unit for the source in which the source or the shielding unit is movable (e.g., by a plunger) would facilitate operation without exposing the source during 212 Pb extraction. Handling the generator inside hot cells or inside glove boxes or bags, or the use of tongs or similar equipment to protect the operator, is an important measure when working with clinically relevant activity levels (e.g., 100 MBq). Automation of the extraction process is considered feasible given that it entails only a surface-washing step and subsequent recovery of the solution.    5 3). † Average of 9. ‡ Average of multiple uses of single generator (n 5 8). ¶ In 6/8 samples, measured radioactivity was below quantification limit after .2 mo (,0.0015% breakthrough; Supplemental Section 1).

220
Rn emanation can be exploited to create a simple and effective generator that produces high-purity 212 Pb without the need for advanced equipment, labor-intensive steps, or hazardous chemicals. Future versions of the presented technology should include simple modifications to shield the source during extraction of the 212 Pb. The generator represents a promising method for efficient 212 Pb production.

DISCLOSURE
Sciencons AS, owned by Roy Larsen, holds intellectual property rights for the presented technology under a patent application. Ruth Li and Vilde Stenberg were industrial PhD students financially supported by the Norwegian Research Council (grants 291228 and 290639) at the time of contributing to the article, at which Vilde Stenberg was also a shareholder at ArtBio AS. Ruth Li is employed at Oncoinvent AS, Vilde Stenberg is employed at ArtBio AS, and Roy Larsen is the chairman of the board of both companies, which use the presented technology for research-and-development projects. Roy Larsen owns stock directly or indirectly in Sciencons AS, Oncoinvent AS, and Art-Bio AS. No other potential conflict of interest relevant to this article was reported. ACKNOWLEDGMENT We thank Marion Masitsa Malenge for the experimental work concerning the radiolabeling of antibodies.

KEY POINTS
QUESTION: Can 212 Pb, intended for radiopharmaceuticals, be produced by a simple generator based on 220 Rn emanation from a 228 Th or 224 Ra source?
PERTINENT FINDINGS: The proposed generator was easy to handle and could routinely be used to produce 212 Pb of high purity, suitable for radiolabeling of antibodies and ligands.
IMPLICATIONS FOR PATIENT CARE: Rapid and efficient production methods such as the one proposed are important for 212 Pb to be available for patients with metastatic cancer.