161Tb-DOTATOC Production Using a Fully Automated Disposable Cassette System: A First Step Toward the Introduction of 161Tb into the Clinic

Visual Abstract

Inr ecent years, peptide receptor radionuclide therapy (PRRT) has emerged as an option in metastatic or nonresectable neuroendocrine neoplasms (NENs) expressing high levels of somatostatin receptor (1,2). DOTATOC and DOTATATE are most commonly used for PRRT in NENs (3). In particular, in the neuroendocrine tumors therapy phase 3 randomized trial (4), 177 Lu-DOTATATE treatment was confirmed as effective in tumor control with only minor side effects. It was approved by the European Medicines Agency in 2017 and the U.S. Food and Drug Administration in 2018 (Lutathera; Advanced Accelerator Applications) for the treatment of well-differentiated gastroenteropancreatic NENs (5,6). Despite its efficacy, studies have since shown partial remission of no more than 50%, with complete response of no more than 18% (4,7,8). The radionuclide 161 Tb (Mean b 2 energy, 154 keV [100%]; half-life, 6.96 d) (9,10) is proposed as a potential alternative to 177 Lu (Mean b 2 energy, 134 keV [100%]; half-life, 6.65 d) (11) because of their similar physical decay characteristics with regard to b 2 -particle emission, suitability for PRRT, and concomitant emission of photons, which can be used for SPECT imaging purposes (Table 1) (12,13). In addition, Tb has similar coordination chemistry to Lu (14); therefore, it can be stably coordinated with a DOTA chelator and respective tumor-targeting peptides, for example, DOTATOC or DOTATATE. 161 Tb is regarded as superior to 177 Lu because it coemits a substantial number of conversion and Auger electrons ($12 e 2 , $37 keV per decay for 161 Tb, $1 e 2 and $1.0 keV per decay for 177 Lu, respectively) (9,11,13), which would be more effective in the treatment of the smallest metastases, as well as single cancer cells (13,(15)(16)(17)(18)(19). 161 Tb production for radiopharmaceutical application was developed and is regularly performed at the Paul Scherrer Institute. It is produced by neutron irradiation of enriched 160 Gd targets. The separated 161 Tb product yields 161 TbCl 3 in a quality suitable for highly specific radiolabeling, which is useful for preclinical applications (13,(18)(19)(20). At a clinical level, phantom studies were performed that demonstrated the feasibility of imaging 161 Tb in high resolution when using low-energy, high-resolution collimators (21); a first-in-humans study was also conducted (22). The next step in the development of this radionuclide toward medical application is its introduction into drug manufacture under good manufacturing practice (GMP) to be able to demonstrate the higher efficacy of 161 Tb-based radiopharmaceuticals than of 177 Lu-labeled counterparts in clinical trials. An approach to demonstrate the sufficient quality of the 161 TbCl 3 solution for clinical purposes was to compare it with the specifications of commercially available no-carrier-added 177 LuCl 3 , because the latter is approved for the preparation of several radiopharmaceuticals for clinical studies (4,23). One purpose of this study was to characterize 161 Tb for clinical use. After this assessment, a protocol for the production of 161 Tb-DOTATOC was developed, conforming to GMP principles, on an automated synthesis module. Tb from the target material, as described previously (20). An approach to demonstrate the high quality of the 161 TbCl 3 product was to compare it with the specifications of commercially available nocarrier-added 177 Lu, approved for the preparation of radiopharmaceuticals for clinical studies, described in the European Pharmacopoeia (Ph. Eur.) monograph (24).

MATERIALS AND
Identity, pH, and Radionuclidic Purity. The identification and radionuclidic purity of 161 Tb were examined by g-spectrometry using a high-purity germanium detector (Canberra), in combination with the InterWinner software package (version 7.1; Itech Instruments; supplemental materials and Supplemental Fig. 1). The choice of g-rays used for a reliable radionuclide identification accounted for possible interferences from other radionuclides that could create artifacts (supplemental materials). The pH of the 161 TbCl 3 solution was assessed by means of pH paper.
Chemical Purity. The chemical purity of the 161 TbCl 3 solution was assessed via inductively coupled plasma-mass spectrometry (Element HR; Thermo Fisher Scientific; supplemental materials). Four batches of 161 Tb were analyzed after the radiochemical purification for contents of Cu, Fe, Pb, Zn, Gd, and Tb, as described in the Ph. Eur. monograph for 177 Lu (24). The purity was also investigated in all 7 161 Tb productions performed during this study by means of apparent molar activity (AMA) determination through hand-operated labeling of the product to DOTATOC (supplemental materials), as described elsewhere (13,20).
Endotoxin Level. Because 161 Tb is considered a radiopharmaceutical precursor, the bacterial endotoxin level needs to be tested. Pyrogens were tested according to Ph. Eur. standards using a chromogenic Limulus amebocyte lysate test (supplemental materials) (25). In the evaluation of the endotoxin level, international units per milliliter were used, considering that the unit used in the Ph. Eur. monograph is international units per volume, indicating the maximum volume to be used for the preparation of a single patient dose, which corresponds to 1 mL of 161 Tb (maximum volume at the end of separation).
Radiochemical Purity (RCP). The RCP of 3 161 Tb batches was assessed by thin-layer chromatography, as described in the Ph. Eur. monograph for 177 Lu (supplemental materials) (24). Quantification of the thin-layer chromatography signals with the indicated software enabled the determination of the area of the peak of 161 Tb in ionic form (R f 5 0.4-0.7), over the total activity. Automated Synthesis of 161 Tb-DOTATOC Ten syntheses of 161 Tb-DOTATOC were performed using a Modular-Lab PharmTracer fully automated cassette synthesis system (Eckert & Ziegler; Fig. 1A), installed in a hot cell so that high activities of the radionuclide could be manipulated with no radiation dose exposure to the operator. For each synthesis, the sterile synthesis cassettes were prepared with the necessary reagents and attached to the Modular-Lab's synthesis module (supplemental materials). A synthesis protocol was adequately adapted and optimized, and the production of 161 Tb-DOTATOC was run by the Modular-Lab PharmTracer software (Modular-Lab SoftPLC; Eckert & Ziegler), which provided a graphical display of each step and the progress of the synthesis together with audit trail as required by GMP (Fig. 1B) (26,27). To ensure sterility, the final product was finally passed through a 0.22-mm filter (Medical Millex-GV Syringe Filter, Hydrophilic Polyvinylidene Fluoride, g-sterilized; Merck Millipore). At the end of the synthesis, the final product activity and the residual radioactivity remaining in the principal components (activity vial, reaction vial, C18 cartridge, and waste) were measured (Isomed Dose Calibrator 2010) (28). Aliquots were withdrawn from the total volume for QC purposes. Further evaluations of the method were performed to investigate the suitability and robustness of the QC. In particular, the linearity, limit of detection, and limit of quantification for both the radiodetector and the UV detector were assessed (Supplemental Fig. 4), together with the setup of a system suitability test (Supplemental Fig. 5).
Gas Chromatography. The ethanol content in the synthesis product was assessed by means of gas chromatography. The quantification of ethanol was based on the acetonitrile internal standard (supplemental materials). The linearity of the gas chromatography method was assessed by linear regression analysis for 6 ethanol concentrations between 1% and 15% (Supplemental Fig. 6).
Sterility and Endotoxin Level. Although the synthesis was not performed under GMP-compliant clean conditions, the pyrogenicity of the product and the integrity of the sterilization filter were evaluated to assess the suitability of the methods and to prove the acceptable quality of 161 Tb-DOTATOC. Pyrogens were tested according to   (25).
To ensure the integrity of the sterilization filter used at the end of the synthesis, a bubble point test was developed. The threshold value of the pressure depends on the matrix of the sterilized solution; therefore, it was assessed for the product in question (supplemental materials) (29). The bubble point test was then performed on the filter used during the synthesis of the batch tested for bacterial endotoxins.
Stability. The stability of the synthesis product, 161 Tb-DOTATOC, was tested in both normal and stress conditions. To establish the stability of the radiolabeled peptide, all synthesized batches of 161 Tb-DOTA-TOC were stored at room temperature and the RCP was assessed 3 and 24 h after synthesis. In addition, 1 batch of DOTATOC radiolabeled with high activity (8.77 GBq) was stored at room temperature for 96 h and the RCP was assessed by means of HPLC every 24 h with the method described earlier. After 96 h at room temperature, the product was incubated at 40 C for an additional 72 h and HPLC analyses were conducted every 24 h.

Tb Production and QC
Multiple productions of 161 Tb were analyzed, compared with the Ph. Eur. monograph for 177 Lu, and found to comply with all requirements (Table 1).
Radionuclidic Purity. The content of the long-lived impurity 160 Tb (half-life, 72.3 d) (30) was less than 0.005% of the total 161 Tb activity at end of bombardment (EOB), as reported previously by our group (20). Three weeks after EOB, the level of 160 Tb was still no more than 0.03% (Supplemental Fig. 1; Supplemental Table 2). Therefore, the specification for radionuclidic purity, which must apply until the end of shelf life, was prudently proposed for 160 Tb at a level of 0.1%. With this level of impurity, the additional radiation dose to the patient was estimated to be about 0.4% (supplemental materials) (31).
Chemical Purity. The content of Cu, Fe, Pb, and Zn measured in the 161 Tb samples was far below the limit for metal impurities reported for 177 Lu (Supplemental Table 3). The Gd content was less than 0.4 ppm. Moreover, 6 of the 7 161 Tb productions were tested for AMA and successfully labeled DOTATOC at 100 MBq/nmol. Even so, the 161 Tb production that allowed labeling of only the lower AMA was sufficiently pure to be used for 161 Tb-DOTATOC synthesis ( Table 2). Thanks to Tb quantification, the specific activity was also calculated and resulted in values greater than 3.5 GBq/mg.
Endotoxin Level. The endotoxin content of the 3 batches tested was less than 1.20, 1.00, and 1.65 IU/mL, respectively, considerably lower than the requirement of less than 175 IU/mL.
RCP. In all productions tested, the peak of 161 Tb in ionic form (R f 5 0.4-0.7) over the total activity ( 161 Tb-diethylenetriaminepentaacetic acid, R f 5 0.9) was above 99.0%, thereby allowing the RCP of 161 Tb to be established as greater than 99.0%.

Automated Synthesis of 161 Tb-DOTATOC
Ten preparations of 161 Tb-DOTATOC were performed and form part of this work. For all syntheses, the 161 Tb-labeled radioactivity at the shelf-life expiration date was in the range from 1 to 7.4 GBq 6 10%, which was the target to obtain a product comparable to a patient dose for 177 Lu-DOTATOC (32). In particular, 5 syntheses produced 161 Tb-DOTATOC with the minimum activity, 4 syntheses produced 161 Tb-DOTATOC with the maximum activity, and 1 synthesis produced 161 Tb-DOTATOC with intermediate activity ( Table 2). The AMA was maintained at a constant of approximately 50 MBq/nmol, varying the peptide amount from 27 mg to a maximum of 200 mg (32). The duration of the process was approximately 45 min, and the total volume of the bulk product at the end of the synthesis was 24 mL, of which 4 mL were intended for QC and 20 mL were intended as final product. The final formulation was established in analogy to that of 177 Lu-DOTATOC (Table 3).
Residual radioactivity was detected in the activity vial, the reaction vial, the C18 cartridge, and the waste (Supplemental Table 4). Unlike the bulk product and initial activity measurements, which were performed with appropriate calibration for each container (28), the residual activity measurements were not comparable to   one another, or to the initial activity, because of the distinct geometries of the samples. Nevertheless, the measurements indicated losses mainly in the waste or because of bound activity on the C18 cartridge. However, a total yield of more than 85% of 161 Tb-DOTATOC, in reference to the initial 161 Tb activity, was determined ( Table 2).

Tb-DOTATOC QC
The synthesis product complied with the established specifications (Table 4).
HPLC. The results of the HPLC analysis of the synthesis products demonstrated that the RCP of 161 Tb-DOTATOC was, on average, approximately 98% at the end of synthesis (n 5 10; 98.2% 6 0.82; Supplemental Table 5), which meets the 95% specification level normally applied to these types of products (Fig. 2) (33,34).
The UV chromatograms showed low levels or complete nonappearance of impurities. The peaks of the unlabeled peptide (DOTATOC), together with those of Tb-DOTATOC, Fe-DOTATOC, and Zn-DOTATOC, established during the development of the HPLC method, were identified during the QC of the synthesis product and considered product peaks (Fig. 3).
Gas Chromatography. The ethanol content in the synthesis product was demonstrated to be less than 10%, which is the acceptable limit for radiopharmaceutical preparation for intravenous injection (35). In particular, the area of the ethanol peak in the analyzed sample of the synthesis product was normalized to the internal standard peak area and then compared with the normalized area of the ethanol peak in the 10% ethanol standard solution. In Figure 4, the ethanol peaks of the standard solution and the synthesis product are visually compared, with the lower ethanol content in the synthesis product displayed.
Sterility and Endotoxin Level. The synthesis resulted in a clear, colorless, particlefree product with a low level of bacterial endotoxins in the batch tested (,6.15 international units/mL). This endotoxin content was compliant with the specification for radiopharmaceutical products defined by the Ph. Eur. monograph (#175 international units/20 mL) (25,33).
Although the sterility of the final product requires further validation, the integrity of the sterilization filter used at the end of the synthesis was successfully proven with a bubble point test. In particular, the bubble point was less than 2.75 bar, as specified for the product matrix (Supplemental Table 6).
Stability. 161 Tb-DOTATOC presented RCP of more than 95% up to 24 h for all batches tested (n 5 9; 97.6% 6 1.0 at 24 h; Supplemental Table 5). In addition, the RCP results of the batch selected for the stability test under stress conditions indicated that 161 Tb-DOTATOC was highly stable at room temperature for up to 4 d (Fig. 5A). When the product was incubated at 40 C, it demonstrated RCP of less than 95% over a period of 48 h, with increased released 161 Tb and radiolysis (Fig. 5B).

DISCUSSION
The aim of this work was to characterize 161 Tb for clinical use, creating a list of parameters that can be the basis for future official specifications and monographic standards. The study compared 177 Lu specifications with 161 Tb features, showing that the latter comply with all requirements reported in the Ph. Eur. monograph for 177 Lu (24). With regard to radionuclidic purity, long-lived 160 Tb (half-life, 72.3 d) (30) was found as an impurity; it is coproduced by the 159 Tb(n,g) 160 Tb nuclear reaction, and the multistep reactions on the residual 159 Tb, 158 Gd, and 157 Gd present as impurities in the target material. However, the content did not exceed 0.005% of the total 161 Tb activity at EOB. Three weeks after EOB, the ingrowth of 160 Tb was still no more than 0.03%. These results were observed in several productions after the irradiation of various amounts of target material with different neutron fluxes. According to previous studies by our group (20), the quality of 161 Tb in terms of radiolabeling yield and AMA was comparable to that of commercially available 177 Lu for 9 d after end of separation. This would prudently set the specifications to a shelf life of 9 d after end of separation and a radionuclidic purity of at least 99.9%. Because the latter would be true until 21 d after EOB, to comply with these specifications, the separation cannot be performed later than 12 d after EOB. However, these assumptions may no longer appear to be true if the target material has a different enrichment level from that used in this study; hence, more research is foreseen to examine this subject in greater detail. In addition, our earlier reports on the radiolabeling yield were supported by the results from this study with regard to 161 Tb chemical purity and RCP. RCP tests showed that more than 99% of 161 Tb was present in the chloride form, which is the expected chemical form after radiochemical separation,  ensuring its direct use for radiolabeling. In terms of chemical impurities, the tested metals complied with the same requirement as stated for 177 Lu. Moreover, only trace amounts of Gd were measured, implying the success of the radiochemical separation from the target material to provide 161 Tb with a quality suitable for clinics. The specific activity resulted in more than 3.5 GBq/mg, which is comparable to what was achieved for commercially available no-carrier-added 177 Lu (36). Because 161 TbCl 3 solution is not intended for direct administration to humans or for use in kit-type products at this stage of development, sterility does not need to be guaranteed (24). However, the product needs to be nonpyrogenic; therefore, pyrogens were tested according to Ph. Eur. standards with a chromogenic Limulus amebocyte lysate test (25), resulting in values below the recommended level.
Taking the preceding observations into consideration, one can see that 161 Tb-labeled radiopharmaceuticals can easily be produced and made GMP-compliant for routine clinical use. The production, including synthesis and QC, of 161 Tb-DOTATOC, the 161 Tb equivalent of the somatostatin analogs currently used with 177 Lu in clinical practice (37,38), was demonstrated. An Eckert & Ziegler cassette synthesis module, with a modified sequence, was applied to radiolabel DOTATOC with levels of 161 Tb radioactivity suitable for a patient dose comparable to that used for 177 Lu-DOTATOC, that is, 7.4 GBq in 20 mL (32). Ten productions were performed, yielding approximately 50 MBq/nmol AMA with up to 9.3 GBq of 161 Tb-DOTATOC produced, which was enough to guarantee the required activity dose for a shelf life of 24 h. Precise activity measurement of 161 Tb, which emits g-radiation mainly with energies below 100 keV, becomes inaccurate with radionuclide dose calibrators used without appropriate calibration factors. An intensive investigation for the calibration of the dose calibrators with the geometries used in the manufacturing process was conducted in collaboration with the certified Institut de Radiophysique and is reported elsewhere (28). The 24 h-shelf life was established to reduce the activity use during the development work, but according to the stability studies, it could easily be extended for up to 4 d. The design of the synthesis system was based on disposable and sterilized flow paths that, together with the final filtration through a 0.22-mm filter, are expected to deliver a pharmaceutically adequate product (33,34). In addition, during the synthesis, 161 Tb-DOTATOC was passed through a C18 cartridge to guarantee the removal of unreacted 161 Tb. However, the final formulation contained diethylenetriaminepentaacetic acid to ensure the coordination of possible traces of released 161 Tb, which is then quickly eliminated via renal excretion, preventing bone uptake of activity after the administration (39). The final product was then tested for RCP and ethanol and endotoxin content, which were determined to be in the acceptable ranges for pharmaceutical preparations. A tendency toward a lower RCP was observed in batches with high activity levels, which may be attributed to the greater activity concentration that caused the peptide to undergo more radiolysis. However, the RCP values for all products were far higher than the criterion for RCP of more than 95%. As a result of these assessments, further purification of the peptide was not required.

CONCLUSION
In the present work, it was demonstrated that 161 Tb shows quality standards comparable to those of 177 Lu, implying its suitability for potential clinical use. In addition, an efficient and robust procedure for the automated production and QC of 161 Tb-DOTATOC was developed using an automated cassette synthesis module and appropriate analytic techniques. The process described was evaluated in accordance with existing guidelines and quality standards. This will form the foundation of monographic standards for this nuclide and further attempts to introduce the use of Tb radioisotopes in clinical applications.

DISCLOSURE
This work was financially supported by the Swiss National Science Foundation (200021_188495) and the NET Research Foundation Petersen Award. No other potential conflict of interest relevant to this article was reported.

ACKNOWLEDGMENTS
We acknowledge Prof. Dr. Jan Rijn Zeevaart for the supervision and arrangement of the neutron irradiations at the South Africa Fundamental Atomic Research Installation facility, as well as Colin C. Hillhouse for technical support in the radiochemical separations. We thank David E. Schmid and Valeria Farsaci for the quality assurance/ QC support and Rolf Hesselmann for regulatory counsel. We also thank Matthias Martin, Dr. Peter Sprung, and Adelheid Fankhauser for the inductively coupled plasma-mass spectrometry analysis and evaluations, as well as Dr. Francesca Borgna for her contribution in the previous work that led to this project. We are grateful to Prof. Dr. Peter Bernhard for his support in the dosimetry calculations.

KEY POINTS
QUESTION: Why is it important to develop a robust production of 161 Tb and derived radiopharmaceuticals for the treatment of NENs?
PERTINENT FINDINGS: The newly developed radionuclide 161 Tb was characterized for clinical use, and fully automated synthesis of 161 Tb-DOTATOC was achieved, showing positive results regarding the safety and quality of the product.
IMPLICATIONS FOR PATIENT CARE: These findings would form the foundation of the introduction of 161 Tb for clinical applications in the treatment of NENs, which could improve the outcome of the therapy because of its conversion and Auger electron emission targeting small metastases and single cancer cells.