On the Separation of Yttrium-90 from High-Level Liquid Waste: Purification to Clinical-Grade Radiochemical Precursor,Clinical Translation in Formulation of 90 Y-DOTATATE Patient Dose

Abstract

Introduction:

The quality control parameters of in-house-produced ⁹⁰Y-Acetate from high-level liquid waste (HLLW) using supported liquid membrane (SLM) technology were validated and compared with the pharmacopeia standard. The radiolabeling of DOTATATE yielding ⁹⁰Y-DOTATATE in acceptable radiochemical purity (RCP), with expected pharmacological behavior in in vivo models, establish the quality of ⁹⁰Y-Acetate. Clinical translation of ⁹⁰Y-Acetate in formulation of ⁹⁰Y-DOTATATE adds support toward its use as clinical-grade radiochemical.

Methods:

Quality control parameters of ⁹⁰Y-Acetate, namely radionuclide purity (RNP), were evaluated using β⁻ spectrometry, γ-spectroscopy, and liquid scintillation counting. RCP and metallic impurities were established using high-performance liquid chromatography and inductively coupled plasma optical emission spectrometry, respectively. The suitability of ⁹⁰Y-Acetate as an active pharmaceutical ingredient radiochemical was ascertained by radiolabeling with DOTATATE. In vivo biodistribution of ⁹⁰Y-DOTATATE was carried out in nude mice bearing AR42J xenografted tumor. Clinical efficacy of ⁹⁰Y-DOTATATE was established after using in patients with large-volume neuroendocrine tumors (NET). Bremsstrahlung imaging was carried out in dual-head gamma camera with a wide energy window setting (100-250 keV).

Results:

In-house-produced ⁹⁰Y-Acetate was clear, colorless, and radioactive concentration (RAC) in the range of 40–50 mCi/mL. RCP was >98%. ⁹⁰Sr content was <0.85 μCi/Ci of ⁹⁰Y. Gross λ content was <0.8 nCi/Ci of ⁹⁰Y and no γ peak was observed. Fe³⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Pb²⁺ contents were <1.7 μg/Ci. The radiolabeling yield (RLY) of ⁹⁰Y-DOTATATE was >94%, RCP was >98%. The in vitro stability of ⁹⁰Y-DOTATATE was up to 72 h postradiolabeling, upon storage at −20°C. Post-therapy (24 h) Bremsstrahlung image of patients with large NET exhibit complete localization of ⁹⁰Y-DOTATATE in tumor region.

Conclusions:

This study demonstrates that the in-house-produced ⁹⁰Y-Acetate from HLLW can be used for the formulation of various therapeutic ⁹⁰Y-based radiopharmaceuticals. Since ⁹⁰Y is an imported radiochemical precursor available at a high cost in India, this study which demonstrates the suitability of indigenously sourced ⁹⁰Y, ideally exemplifies the recovery of “wealth from waste.”

The Clinical Trial Registration number: (P17/FEB/2019).

Introduction

Yttrium-90, a pseudo-lanthanide and a pure β⁻ emitter, with a nuclear decay characteristics (T_1/2 of 64.1 h and β⁻ _max of 2.27 MeV) decaying to stable ⁹⁰Zr, is an isotope of choice in therapeutic nuclear medicine.¹ The high-energy β⁻ emission, particularly makes it a favorable option in the treatment of large-volume cancers. The absence of an imageable gamma photon in ⁹⁰Y is compensated in most cases by the availability of a matched pair of diagnostic radiopharmaceutical based on the PET isotope Gallium-68. The use of the ⁶⁸Ga-⁹⁰Y matched pair in a theranostic setting is well documented in literature. The thrust on development of ⁹⁰Y-based radiopharmaceuticals lies in its uses for therapeutic modalities such as peptide receptor radionuclide therapy (PRRT), radioimmunotherapy (RIT) of large-volume tumors, and treatment of hepatocellular carcinoma.²

However, the challenges posed in envisaging ⁹⁰Y as a therapeutic radioisotope, are its availability and its purity for clinical use. The possibility of production of ⁹⁰Y from ⁸⁹Y using the direct thermal neutron activation, nγ route, in medium-flux reactors, is not a feasible option due to the low cross-section (1.28 barns) of ⁸⁹Y, which results in low specific activity.³ Therefore, for targeted radiotherapeutic applications, such as PRRT and RIT, the ⁹⁰Y should be carrier free and have a specific activity of 2.01 × 10⁴ GBq/mg.⁴ Toward this, several methods have been adopted worldwide for development of generators to separate the ⁹⁰Y daughter from its long-lived parent ⁹⁰Sr, with which it remains in secular equilibrium. Such generators, which aim at separation of the daughter ⁹⁰Y in purity, equivalent to clinical standards, are based on electrochemical separation,⁵ precipitation,⁶ extraction,⁷ ion exchange chromatography^8,9-supported liquid membrane (SLM) separation¹⁰ etc.

In most of the cases the ⁹⁰Y has been made available in the ⁹⁰YCl₃ form and separated from long-lived ⁹⁰Sr available at high-level liquid waste (HLLW). The most critical requirement is the purification of ⁹⁰Y to the clinical grade form to enable proper radiolabeling with various peptides and monoclonal antibodies (MoAbs) for clinical translation in patients. However, getting clinical-grade ⁹⁰Y with metal contents, namely Fe (< 5 μg/Ci), Pb (< 10 μg/Ci) and Cu, Zn, Al, and Cd (< 30 μg/Ci)^11,12 constitutes a challenging task. However, RNP of ⁹⁰Y solution plays a critical role in determining the final radiolabeling yield. The level of the parent, ⁹⁰Sr (which is β⁻ emitter) in ⁹⁰Y solution is of utmost importance as it is a bone seeker with high physical T_1/2 of 28.8 years. As per the European pharmacopeia the ⁹⁰Sr content should be ≤2.5 μCi/Ci of ⁹⁰Y solution.^13,14

Apart from β⁻ impurities, the levels of gross γ and α contents in ⁹⁰Y solution arising from the presence of various long-lived radio-actinides and fission products, present in HLLW should be very low. The gross γ and α content in a clinical-grade ⁹⁰Y solution should be <0.001% and ≤1 × 10⁻⁷%, respectively.^15,16

Radiolabeling of various peptides and MoAbs using clinical-grade ⁹⁰Y is possible, when the extracted clinical-grade ⁹⁰Y exist in single chemical species. Essentially, the radiochemical purity (RCP) of the extracted ⁹⁰Y solution should be ≥98%. Apart from all these, the clinical-grade ⁹⁰Y solution should be sterile and apyrogenic.

Toward this goal, the authors have extracted clinical-grade ⁹⁰Y in ⁹⁰Y-Acetate form using a SLM technique.¹⁰ The quality control parameters of ⁹⁰Y-Acetate were thoroughly validated and compared with the pharmacopeia standard. Regulatory approval by the Radiopharmaceutical Committee has been obtained for ⁹⁰Y-Acetate for its intended use as a radiochemical precursor in formulation of therapeutic radiopharmaceuticals for use in human patients. The DOTATATE was radiolabeled with ⁹⁰Y-Acetate, yielding the product with acceptable clarity, pH, RCP, RLY, RAC, and endotoxin limit (EL).

Furthermore, the in vitro and serum stability of ⁹⁰Y-DOTATATE were established. The in vitro studies, namely cell binding, cell internalization, and competition assay studies, of ⁹⁰Y-DOTATATE, were carried out on pancreatic adenocarcinoma AR42J cell-line-expressing somatostatin receptor-2 (SSTR-2). The in vivo pharmacological behavior of ⁹⁰Y-DOTATATE was established by biodistribution studies in male athymic nude mice carrying AR42J pancreatic adenocarcinoma xenograft tumors.¹⁷

This ⁹⁰Y-Acetate has been utilized in the formulation of patient doses of ⁹⁰Y-DOTATATE and subsequently used in the treatment of large, progressive, disseminated, metastatic, and neuroendocrine tumors in patients.

The consistency in the quality control parameters, in vitro stability, pharmacokinetic studies in suitable cell lines, and in vivo biodistribution in tumor-bearing mice of ⁹⁰Y-DOTATATE, added support to its clinical translation in human patients following the approval by the Regulatory Committee.

Materials and Methods

Separation and purification of ⁹⁰Sr from HLLW

Separation of ⁹⁰Sr: HLLW from reprocessing plant was put through three cycles of solvent extraction process. In first cycle, depletion of residual uranium and plutonium from the waste was carried out using the plutonium uranium reduction extraction (PUREX) solvent that is 30% Tri-n-butyl phosphate (TBP) in n-dodecane (∼ 93% C12, sp. gr. = 0.751, refractive index = 1.42, Transware Chemia, Germany). Uranium (U) and plutonium (Pu) from organic phase are stripped in aqueous phase using 0.01 M HNO₃ and sent back for reprocessing. The U/Pu lean raffinate from first cycle was put through a second solvent extraction cycle using domestically synthesized 0.03 M 1,3-dioctyloxycalix[4]arene-crown-6 (CC6) in 30% isodecanol/n-dodecane for selective recovery of ¹³⁷Cs. Raffinate from ¹³⁷Cs recovery cycle is subjected to N,N,N′,N′,-tetra (2-ethylhexyl) diglycolamide (TEHDGA) in isodecanol/n-dodecane.

In this third cycle, entire actinides/lanthanides and ⁹⁰Sr are extracted quantitatively in organic phase leaving raffinate stream amenable to direct dilution and dispersal. Stripping of the organic phase using dilute nitric acid generates aqueous stream rich in ⁹⁰Sr activity. This aqueous phase generated from stripping also contains minor actinides/lanthanides and traces of ¹³⁷Cs activity.¹⁸

Purification of ⁹⁰Sr: In the first step, trace impurities of ¹³⁷Cs was removed using granulated ammonium phosphomolybdate column. In the second step, α emitters, namely minor actinides, lanthanides, and traces of U and Pu, were removed by extraction chromatography using Truex solvent {0.2 M octyl(phenyl)-N,N-diisobutylcarbamoylmethyl phosphine oxide (CMPO, synthesized in-house) and 1.2 M TBP in n-dodecane}. In the third step, raffinate containing ⁹⁰Sr in 3–4 M HNO₃ (Sigma-Aldrich) was subjected to radiostrontium extraction process using 4,4′(5′)-bis(tert-butylcyclohexano)-18-crown-6 (Fluka Chemie, Switzerland) in isodecanol/n-dodecane. Finally, ⁹⁰Sr from the organic phase was stripped quantitatively using 0.01 M HNO₃ (Sigma-Aldrich).

In the fourth step, the stripped ⁹⁰Sr (obtained after the third step of extraction process) was passed through a glass column containing polymeric absorbent that is XAD-7 (Supelco) to remove dissolved organic material if any. The resultant solution was evaporated to get concentrated ⁹⁰Sr using a customized in-house-designed oven with a vent connected to fumehood. The ⁹⁰Sr obtained from the above feed solution was further purified to remove small impurities and concentrated by employing radiochemical precipitation method.

About 10 mg of iron and 10 mg of strontium were added as carriers to the impure ⁹⁰Sr(NO₃)₂ solution. Iron was precipitated as hydroxide along with other impurities by the addition of ammonia solution drop-wise until the pH of the supernatant reached ∼9.4. After centrifuging, the supernatant was collected in a separate vial. The precipitate was washed once with 5% ammonia solution and the wash was collected in the same vial. The hydroxide precipitate was discarded. Sr(OH)₂ solution thus obtained was acidified and precipitated as carbonate using 5% solution of Na₂CO₃. This resulted in >80% recovery of ⁹⁰Sr.

The final SrCO₃ precipitate was separated after centrifugation_, further washed with water, and dissolved in minimum volume of 2 M HNO₃, (Sigma-Aldrich), while the pH was adjusted between 1 and 2. Purified ⁹⁰Sr(NO₃)₂ solution was allowed to reach equilibrium, for extracting ⁹⁰Y employing the two-stage SLM generator. After this stage of purification, the ⁹⁰Y was extracted from ⁹⁰Sr(NO₃)₂ by employing the two-stage SLM generator. The gross α levels in the extracted ⁹⁰Y was found to be 10⁻⁸ Ci/1.0 Ci of ⁹⁰Y.¹⁸

Additional purification of ⁹⁰Sr

This additional purification of ⁹⁰Sr was carried out to reduce the gross α levels in clinical-grade ⁹⁰Y-Acetate radiochemical from 10⁻⁸ Ci/Ci of ⁹⁰Y to ≤10⁻⁹ Ci/Ci of ⁹⁰Y. The parent ⁹⁰Sr(NO₃)₂ solution obtained after radiochemical precipitation was kept in the feed chamber and allowed to transport to the receiver chamber containing 4M HNO₃ (Sigma-Aldrich) through KSM-17-impregnated PolytetraFluoro ethylene (PTFE) membrane (Merck) in the first stage of separation. The 4 M HNO₃ used in the receiver chamber was replaced at intervals of 8 h and discarded.

This receiving chamber was filled with fresh 4 M HNO₃ and the process was repeated for six times keeping the parent ⁹⁰Sr(NO₃)₂ in the feed compartment intact. The purified ⁹⁰Sr(NO₃)₂ solution from the feed compartment thus obtained was allowed to grow the β⁻ activity for 23–25 d and further used for extraction of ⁹⁰Y based on the SLM technique. These steps were carried out to reduce the gross α levels of clinical-grade ⁹⁰Y-Acetate from 10⁻⁸ Ci/1.0 Ci of ⁹⁰Y to <10⁻⁹ Ci/1.0 Ci of ⁹⁰Y.¹⁸

Separation of clinical-grade ⁹⁰Y in ⁹⁰Y-Acetate form using the two-stage SLM generator

A two-stage SLM-based generator system developed in-house and used for the separation of carrier-free ⁹⁰Y based on the solvent extraction properties of two ligands, namely 2-ethylhexyl 2-ethylhexyl phosphonic acid (KSM-17; BARC) and octyl phenyl-N,N-diisobutylcarbamoyl methyl phosphine oxide (CMPO; BARC) under optimum conditions. The system was operated in sequential modes with each borosilicate glass cell having 5 mL capacity (BARC).

In the first stage, the equilibrium mixture of ⁹⁰Sr and ⁹⁰Y adjusted to pH 1-2, which was used in the feed compartment, and the receiver compartment contained 4 M HNO₃. KSM-17-based SLM was used for selective transport of ⁹⁰Y to the receiver phase in about 4 h. The product from this stage was taken out and placed in the feed compartment of the second stage, whereas the ⁹⁰Y depleted lean ⁹⁰Sr left out in the feed compartment of the first stage was transferred back in the feed reservoir for next cycle. CMPO-based SLM was used for transport of ⁹⁰Y in the second stage where 1 M CH₃COOH (Sigma-Aldrich) was used as receiver phase¹⁰¹⁸.

Physicochemical and biological quality control of ⁹⁰Y-Acetate

The RCP was evaluated by thin-layer chromatography (TLC) (RayTest) using 60A^o silica gel plastic TLC plates (Merck, Germany) and 0.1 M sodium citrate buffer, pH-5.0 as eluent. High-performance liquid chromatography (HPLC; Knauer) analyses were carried out using RP18 column, (isocratic mode) with eluent as 0.1%TFA in water/acetonitrile: 80/20.

RNP, namely gross γ, was determined by high-purity germanium (HPGe) coupled to 4 and 64 k multiple-channel analyzer (MCA) for 24 and 2 h, respectively (Baltic Scientific Instruments, Russia and ITECH Instruments). Gross α was quantified by ZnS(Ag) method and solid-state track detector CR-39 (Nucleonix). ⁹⁰Sr content was quantified both by extraction paper chromatography (EPC) and β⁻ spectrometry (Nucleonix).

Metallic impurities (Fe, Pb, Cu, Zn, and Cd) were determined by inductively coupled plasma optical emission spectrometry (ICP-OES) (Thermo Fisher Scientific). Phosphonic acid-based impurities generated from radiolytic damage of KSM-17 and 0.8 M CMPO-coated PTFE membrane was analyzed by 600 MHz ³¹P nuclear magnetic resonance (³¹P-NMR) (JEOL) in decayed ⁹⁰Y-Acetate samples. Since the ⁹⁰Sr/⁹⁰Y-equilibrated feed mixtures in the first and second stages were 0.02 and 4 M HNO₃ respectively, determination of NO₃ ⁻ (Nitrate ion) concentration was carried out by ion chromatography (ACD; BARC). EL was quantified by the Gel-clot BET assay (Charles River). Sterility test was performed by the direct inoculation method (Himedia).

Radiolabeling of DOTATATE with ⁹⁰Y-Acetate and its quality control

Carrier-free, clinical-grade ⁹⁰Y-Acetate was sourced from a two-stage⁹⁰Sr/⁹⁰Y generator system based on SLM technology. The formulation for single patient dose (85–90 mCi) ⁹⁰Y-DOTATATE was carried out using in-house-produced ⁹⁰Y-Acetate, 0.2 N ammonium acetate buffer (pH: 5.5) (Sigma-Aldrich), and GMP grade DOTATATE acetate (ABX Biochemicals). The reaction mixture was incubated at 95°C for 35–40 min at pH ∼4.0–4.5. On cooling, 60 mg of 2, 5-dihydroxybenzoic acid (Sigma-Aldrich) per mL of saline was added. The resultant ⁹⁰Y-DOTATATE was diluted with sterile, pyrogen-free saline (Nirlife) so as the radioactive concentration (RAC) was maintained between 8 and 10 mCi/mL and filtered using sterile 0.22 μm poly ether sulfone membrane syringe filter (Merck).

RCP was assessed by TLC (60A^o-silica-gel, 0.1 M sodium citrate buffer: pH-5.0) (RayTest) and HPLC (Knauer) by two different gradient modes, namely steep and shallow (Solvent: 0.1% TFA in H₂O and CH₃CN, steep gradient method: 0–5 min 95% water, 5–10 min 95% to 0% water, 10–15 min 0% water, 15–20 min 0% to 95% water, and 20–25 min 95% water. shallow gradient method: 0–4 min 95% water, 4–15 min 95% to 5% water, 15–20 min 5% water, 20–25 min 5% to 95% water, and 25–30 min 95% water) using RP18 column coupled with NaI (Tl) and UV-254 nm detector maintaining a flow rate of 1.0 mL/min. The EL was quantified by gel-clot BET assay and sterility test was done by direct inoculation. In vitro and serum stability of the product on storage at −20°C was evaluated by TLC/HPLC at 24, 48, and 72 h postradiolabeling.

In vitro studies of ⁹⁰Y-DOTATATE

Cell binding

Pancreatic carcinoma cell-line AR42J (CLS Cell Lines Service; Eppelheim)-expressing SSTR-2 (Somatostatin receptor, subtype 2), used for the in vitro evaluation, was grown in IMDM (Gibco, Thermo Fisher Scientific) with 10% FBS in 5% CO₂ at 37°C (Thermo Fisher Scientific). In vitro cell-binding studies were performed by incubating AR42J cells in 1mL of internalization buffer (IMDM, 0.2% BSA) containing radioligand (∼5 × 10^–12 mol peptide) for 15, 30, 60, and 120 min followed by washing with PBS. Quantification of radioactivity was performed in β⁻ counter (Nucleonix). For membrane receptor-binding assay, AR42J cell homogenates were incubated above time points, followed by washing with PBS. MCF7 cell (NCCS cell repository) was used as negative control for SSTR2 expression.

Cell internalization

Pancreatic carcinoma cell-line AR42J-expressing SSTR-2, used for the in vitro evaluation, was grown in IMDM with 10% FBS in 5% CO₂ at 37°C. In vitro cell-binding studies were performed by incubating AR42J cells in 1 mL of internalization buffer (IMDM, 0.2% BSA) containing radioligand (∼5 × 10^–12 mol peptide) for 15, 30, 60, and 120 min followed by washing with PBS. Quantification of radioactivity was performed in β⁻ counter.

Competition assay

The receptor binding affinity and nonspecific binding was determined by preincubation of SSTR-2-expressing AR42J cells with cold DOTATATE (1 nM) followed by incubation with ⁹⁰Y-DOTATATE (0.5 and 1 pM) for 120 min. Competitive binding of the cold conjugate resulted in complete inhibition of cell surface receptor binding as well as internalization of the radioconjugate as studied using live AR42J cells.

In vivo biodistribution studies of ⁹⁰Y-DOTATATE

The degree of receptor-mediated uptake was determined by biodistribution studies of the radioconjugate in male athymic nude mice (Vivo Biotech). The nude mice were injected with pancreatic neuroendocrine carcinoma cell-line AR42J (2 × 10⁶ cells/mice) at the proximal flank region of the mice, and tumor was allowed to grow to 1 cm³ volume.

The radioconjugate, ⁹⁰Y-DOTATATE (50-100 μCi/mice), was injected in xenografted mice through the tail vein. The biodistribution studies were done at 6, 24, 48, and 72 h postinjection, for evaluation of the ⁹⁰Y-DOTATATE. Before sacrificing, these mice were imaged on a gamma camera (dual-head Siemens E-cam) for Bremsstrahlung radiation to study the uptake in the tumor. PET/CT imaging was performed in mice by injecting a higher dose of ⁹⁰Y-DOTATATE due to the lesser fraction positron emission after administering lasix for bladder clearance and under isoflourane sedation using Philips Gemini TF 16-slice camera, Netherlands.

Clinical studies of ⁹⁰Y-DOTATATE

Patients (n = 12) having large neuroendocrine tumors (NET) with gross liver involvement showing good tumor uptake on diagnostic ⁶⁸Ga-DOTATATE study were selected for treatment with ⁹⁰Y-DOTATATE. The patients underwent renogram studies to check the renal status along with other blood investigations before therapy. Patients were admitted in the ward and cationic amino acid infusion was started 4 h before the therapy. ⁹⁰Y-DOTATATE (85–90 mCi per patient) was administered followed by another 4 h of amino acid infusion.

Twenty two to 24 h post-therapy, the patients were imaged on a dual-head Siemens E-cam γ-camera with the help of Bremsstrahlung radiation to study the uptake in the tumor. Bremsstrahlung imaging was performed with wide energy window setting (100–250 keV). Post-therapy, a regional PET/CT scan was carried out on a Philips Gemini TF 16-slice camera for 30 min to study imaging characteristics of ⁹⁰Y-DOTATATE with low positron flux of ⁹⁰Y.

Results

In this work, extensive evaluation of quality control parameters of ⁹⁰Y-Acetate samples (n = 44, batch size: 200 mCi) with RAC of 40–50 mCi/mL were analyzed and presented. Further radiolabeling and quality control study data were presented for ⁹⁰Y-DOTATATE (n = 24) formulated using indigenously sourced ⁹⁰Y-Acetate.

RNP of ⁹⁰Y-Acetate

In the indigenously sourced 44 batches of ⁹⁰Y-Acetate from HLLW, the β⁻ content arising due to the presence of ⁹⁰Sr determined by EPC method were found to be 0.83 ± 0.08 μCi/Ci of ⁹⁰Y, which were below the permissible limits of 2.5 μCi/Ci of ⁹⁰Y. β⁻ spectrum of ⁹⁰Y-Acetate solution shows single peak with E_max of 2.28 MeV (Fig. 1A), whereas β⁻ spectrum of decayed ⁹⁰Y-Acetate samples (post 65 d of extraction) does not exhibit any prominent peak (Fig. 1B). For validation of our β⁻ spectroscopy method, the β⁻ spectrum (Fig. 1C) of ⁹⁰Sr/⁹⁰Y equilibrated mixture shows two peaks with E_max of 0.546 and 2.28 MeV corresponding to ⁹⁰Sr and ⁹⁰Y, respectively.

FIG. 1.

Beta (β⁻) Spectrum of (A) ⁹⁰Y-Acetate Solution (40 nCi/50 μL); (B) ⁹⁰Y-Acetate Solution (65 d postextraction); (C) ⁹⁰Sr/⁹⁰Y Equilibrium Mixture. Color images are available online.

The presence of any γ ray-emitting radionuclides was analyzed in all the produced batches of ⁹⁰Y-Acetate (n = 44). The γ spectrum recorded for 2 h using 64 k MCA channel coupled to HPGe detector shows large Bremsstrahlung spectrum characteristics of a pure β⁻ emitter. Another 511 keV peak was observed over large Bremsstrahlung spectrum, which is attributed to the internal pair production for the branching ratio for the 0⁺ −0⁺ transition of ⁹⁰Zr ¹⁹ (Fig. 2A). The γ spectrum of ⁹⁰Y-Acetate sample of 24 h using 4 k MCA channel exhibits background X ray (< 100 keV), ¹³⁷Cs (1460 keV), and ⁴⁰K (661 keV), apart from the 511 keV peak of 0⁺ −0⁺ transition of ⁹⁰Zr (Fig. 2B). In Figure 2B, the large Bremsstrahlung peak characteristic of ⁹⁰Y diminished using shielding for the detector made from alloy of lead, cadmium, and copper.

FIG. 2.

Gamma (γ) spectrum of (A) ⁹⁰Y-Acetate counted for 2 h using 64 k MCA (B) ⁹⁰Y-Acetate counted for 24 h using 4 k MCA. MCA, multiple channel analyzer. Color images are available online.

The gross α contents in all the ⁹⁰Y-Acetate batches (n = 44) arising from various actinides in HLLW were 0.78 ± 0.26 nCi/Ci of ⁹⁰Y, determined using liquid scintillation counting system. The results of gross α contents in ⁹⁰Y-Acetate counted by liquid scintillator were correlated with the results obtained from ZnS(Ag)-based scintillation counter and were found to be 0.80 ± 0.14 nCi/Ci of ⁹⁰Y. The gross α contents in our ⁹⁰Y-Acetate samples estimated by both the methods were much below the permissible limits of 1 nCi/Ci of ⁹⁰Y.^20,21

The RCP of ⁹⁰Y-Acetate

The RCP of ⁹⁰Y-Acetate samples (n = 44) estimated by radio-TLC method using two different mobile phases that is 0.1 M sodium citrate buffer (pH: 5.0) and 0.1 M sodium citrate/1 M HCl (97/3 v/v) (pH: 4.5) were 98.9% ± 0.66% (R_f between 0.8 and 0.9) and 98.9% ± 0.73% (R_f between 1.0 and 1.1), respectively (Fig. 3A, B). The RCP derived by radio-HPLC was 98.9% ± 0.55% with R_t between 3 and 5 min (Fig. 3C). The RCP of all the batches of ⁹⁰Y-Acetate samples derived by TLC and HPLC method was above the specified limits of >98%.

FIG. 3.

Radio-Thin-Layer Chromatogram of ⁹⁰Y-Acetate in (A) 0.1 M Sodium Citrate Buffer, pH: 5.0 (R_f: 0.80); (B) 0.1 M Sodium Citrate/1 M HCl, 97/3 (v/v), pH: 4.5 (R_f: 1.08); (C) Radio-High-Performance Liquid Chromatogram of ⁹⁰Y-Acetate (R_t: 4.59 min). Color images are available online.

Metallic impurities in ⁹⁰Y-Acetate

In all the batches of ⁹⁰Y-Acetate (n = 44) the levels of Fe³⁺, Cu²⁺, Zn²⁺, Cd²⁺, and Pb²⁺ were (1.66 ± 1.15), (0.83 ± 0.60), (0.91 ± 0.70), (0.60 ± 0.40), and (1.39 ± 1.09) μg/Ci of ⁹⁰Y, respectively. The observed Fe³⁺, Cu²⁺/Zn²⁺/Cd²⁺ and Pb²⁺ levels in ⁹⁰Y-Acetate batches were much below the specified limit of <5 μg/Ci, <30 μg/Ci, and <10 μg/Ci, respectively.

Nitrate ions, phosphorus, and stable ⁹⁰Zr contents in ⁹⁰Y-Acetate

The concentration of nitrate ions, which could be transported through CMPO-impregnated PTFE membrane in ⁹⁰Y-Acetate solutions (n = 44) was (0.94 ± 0.52) %. With this concentration, the amount of nitrate ions when calculated during single PRRT, with a patient dose of 100 mCi using ⁹⁰Y-DOTATATE, works out to be 1.25 mg/kg of body weight. These levels of nitrate ions in ⁹⁰Y-labeled peptides are considered to be nontoxic for patients undergoing PRRT since the acceptable daily intake of nitrate ions is 3.7 mg/kg.²²

The absence of any peak in proton-decoupled ³¹P-NMR of decayed ⁹⁰Y-Acetate samples confirms the absence of any phosphonic acid-based compounds (Fig. 4A). The single peak (proton) in ¹H-NMR of the decayed ⁹⁰Y-Acetate samples is due to the presence of 1 M CH₃COOH (Fig. 4B). As a confirmatory measure, the ³¹P-NMR of KSM-17 and 0.8M CMPO solution were recorded and the single peak observed for Phosphorus-31 could not be seen in the proton-decoupled ³¹P-NMR of decayed ⁹⁰Y-Acetate samples. This eliminates the possible presence of any radiolytically degraded phosphorus-based polymers in ⁹⁰Y-Acetate samples (Fig. 4C, D).

FIG. 4.

Proton-decoupled ³¹P-NMR spectrum of (A) ⁹⁰Y-Acetate (65 d postextraction); (B) ¹H-NMR spectrum of ⁹⁰Y-Acetate (65 d postextraction); (C) ³¹P-NMR spectrum of KSM-17; (D) ³¹P-NMR spectrum of CMPO (0.8 M) in n-dodecane. Color images are available online.

The ⁹⁰Zr content observed in the ⁹⁰Y-Acetate (n = 44) was 0.30 ± 0.17 μg/Ci of ⁹⁰Y. This concentration of stable ⁹⁰Zr in ⁹⁰Y-Acetate, when calculated in a single patient dose of 100 mCi undergoing PRRT using ⁹⁰Y-DOTATATE, corresponds to 0.4 ng/kg of body weight. These levels of stable ⁹⁰Zr in ⁹⁰Y-labeled peptides are nontoxic, since the acceptable limit for annual intake of stable ⁹⁰Zr through food is 18 mg/kg of the body weight.²³

EL and sterility of ⁹⁰Y-Acetate

The EL for all the batches of indigenously sourced ⁹⁰Y-Acetate (n = 44) was found to be <3 EU/mL, which is below the specified limit of <5 EU/mL. The ⁹⁰Y-Acetate was found to be sterile.

The sterility test by direct inoculation method was validated by setting up positive cultures using microorganisms, namely Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, and Candida albicans. Luxuriant growths in media bottles were observed containing inoculums of microorganisms and ⁹⁰Y-Acetate within 3 d of incubation.

Table 1 demonstrates the quality control results of in-house-produced ⁹⁰Y-Acetate.

Table 1.

Quality Control Results of ⁹⁰Y-Acetate

Description		Specifications		Observed results
Characteristics of the Solution		Clear, Colorless, and Free of particulate matter		Complies
pH		2.2–2.5		∼2.3
Radioactive concentration		40–50 mCi/mL		∼43–46 mCi/mL
γ Radionuclide Impurity (Gross)		<0.001%		Not significant γ peak was observed, except Bremsstrahlung peak (Characteristics of ⁹⁰Y)
β ⁻ Radionuclide Impurity (⁹⁰Sr content - at calibration date)		EPC	≤ 2.5 μCi ⁹⁰Sr/1.0 Ci ⁹⁰Y	0.83 ± 0.08 μCi/1.0 Ci of ⁹⁰Y
		β ⁻ Spectroscopy	≤ 2.5 μCi ⁹⁰Sr/1.0 Ci ⁹⁰Y	Single peak with E_max of 2.28 MeV was observed. (Characteristics of ⁹⁰Y)
α Radionuclide Impurity (Gross)		Liquid scintillation counting	≤ 1.0 nCi/1.0 Ci of ⁹⁰Y	0.78 ± 0.26 nCi/1.0 Ci of ⁹⁰Y
α Radionuclide Impurity (Gross)		ZnS(Ag) based scintillation counting	≤ 1.0 nCi/1.0 Ci of ⁹⁰Y	0.80 ± 0.14 nCi/1.0 Ci of ⁹⁰Y
Radiochemical Purity		TLC Method	≥ 98% (⁹⁰Y as Y³⁺)	98.9% ± 0.66%
Radiochemical Purity		HPLC Method	≥ 98% (⁹⁰Y as Y³⁺)	98.9% ± 0.55%
Metallic Impurities (μg/Ci)	Fe	<5		1.66 ± 1.15
	Pb	<10		1.39 ± 1.09
	Cu	<30		0.83 ± 0.60
	Zn	<30		0.91 ± 0.70
	Cd	<30		0.60 ± 0.40
Endotoxin limit		<5 EU/mL		< 3 EU/mL
Sterility Test (Post facto)		Absence of any growth on 14 d of incubation		Complies

EPC, extraction paper chromatography; EU, endotoxin unit.

Regulatory approval

Regulatory clearance from DAE's Radiopharmaceutical Committee was obtained for ⁹⁰Y-acetate as a clinical-grade radiochemical for use in formulation of ⁹⁰Y-based radiopharmaceuticals

Radiolabeling and quality control of ⁹⁰Y-DOTATATE

Using clinical-grade ⁹⁰Y-Acetate, a single patient dose of 95–100 mCi of ⁹⁰Y-DOTATATE was formulated (n = 24) with RLY 94.07% ± 2.68%_. In-house-produced ⁹⁰Y-DOTATATE was found to be clear and of pale yellow color, whereas pH was in the range of 4.0–6.0. The RAC was observed to be in the range of 8–10 mCi/mL. The maximum RLY was observed when ⁹⁰Y to DOTATATE used were in the molar ratios of 1: 25. Table 2 shows the decreased RLY, when molar ratios of ⁹⁰Y to DOTATATE were reduced to 20, 15, 10, and 5.

Table 2.

Influence of Metal/Ligand Ratio on RLY of ⁹⁰Y-DOTATATE

Metal (⁹⁰Y): Ligand (DOTATATE) Ratio	RLY (%)
1:25	94.07 ± 2.68
1:20	78.34 ± 1.01
1:15	63.72 ± 1.68
1:10	62.50 ± 0.98
1:5	33.26 ± 1.26

RLY, radiolabeling yield.

The RC Purities of ⁹⁰Y-DOTATATE (n = 24) estimated by radio-TLC and radio-HPLC were 98.74% ± 0.49% with R_f between 0.00 and 0.10 (Fig. 5A) and 99.11% ± 0.55% with R_t between 10 and 12 min (Fig. 5B), respectively. The EL of ⁹⁰Y-DOTATATE was <6 EU/mL and the produced radiopharmaceutical was found to be sterile.

FIG. 5.

Radio-Thin-Layer chromatogram of (A) ⁹⁰Y-DOTATATE in 0.1 M Sodium Citrate Buffer, pH: 5.0 (R_f: 0.06); (B) Radio-High-Performance Liquid Chromatogram of ⁹⁰Y-DOTATATE (R_t: 11.27 min); (C) Radio-Thin-Layer chromatogram of ⁹⁰Y-DOTATATE and ⁹⁰Y-Acetate in 0.1M Sodium Citrate Buffer, pH: 5.0 (R_f: 0.04 and 0.92); (D) Radio-High-Performance Liquid Chromatogram of ⁹⁰Y-DOTATATE and ⁹⁰Y-Acetate (R_t: 11.3 and 3.35 min). Color images are available online.

For validating the radio-TLC and radio-HPLC method, the ⁹⁰Y-DOTATATE was spiked with a known concentration of ⁹⁰Y-Acetate. Radio-TLC chromatogram (Fig. 5C) exhibits two peaks with R_f 0.92 and 0.04 corresponding to ⁹⁰Y-Acetate and ⁹⁰Y-DOTATATE, respectively, whereas radio-HPLC chromatogram (Fig. 5D) exhibits two peaks with R_t 3.35 and 11.3 min corresponding to ⁹⁰Y-Acetate and ⁹⁰Y-DOTATATE.

Stability of ⁹⁰Y-DOTATATE

In vitro (radio-HPLC, Fig. 6A) stability of the ⁹⁰Y-DOTATATE was found to be 98.70% ± 0.53% up to 72 h upon storage at −20°C using 60 mg/mL of gentisic acid as the stabilizer. Radio-TLC chromatogram (Fig. 6B) ascertains the in vitro serum stability of ⁹⁰Y-DOTATATE with RCP of 98.62% ± 0.47% upon 24 h postincubation (37°C) and further storage for 24 h at −20°C.

FIG. 6.

(A) Radio-High-Performance Liquid Chromatogram of ⁹⁰Y-DOTATATE (R_t: 10.35 min) with stabilizer at 72 h postradiolabeling on storage at −20°C; (B) Radio-Thin-Layer chromatogram of supernatant fraction of ⁹⁰Y-DOTATATE at 24 h postincubation with serum. Color images are available online.

Table 3 demonstrates the physicochemical and biological quality control results of the produced ⁹⁰Y-DOTATATE.

Table 3.

Quality Control Results of ⁹⁰Y-DOTATATE

Description	Specifications		Observed results
Characteristics of the solution	Clear, Pale yellow color and Free of particulate matter		Complies
pH	4.0–6.0		∼4.5–5.5
Radioactive concentration	8–10 mCi/mL		∼8.5–9.0 mCi/mL
Radiolabeling Yield	90%		94.07% ± 2.68%_.
Radiochemical purity (RCP)	TLC Method	≥ 95% as ⁹⁰Y-DOTATATE	98.74% ± 0.49%
Radiochemical purity (RCP)	HPLC Method	≥ 95% as ⁹⁰Y-DOTATATE	99.11% ± 0.55%
Endotoxin limit	< 25 EU/mL		<6 EU/mL
sterility test (Post facto)	Absence of any growth on 14 d of incubation		Complies
In vitro stability	HPLC method	RCP shall be ≥95%, 24 h postformulation on storage at −20°C.	98.70% ± 0.53%
Serum stability	TLC method	RCP shall be ≥95%, upon 24 h postincubation (37°C) and further storage for 24 h at −20°C.	98.62% ± 0.47%

HPLC, high-performance liquid chromatography; TLC, thin-layer chromatography.

In vitro cell binding and cell internalization of ⁹⁰Y-DOTATATE

In vitro cell-binding studies carried out in AR42J cells showed a rapid and specific binding of 25% ± 0.1% with ⁹⁰Y-DOTATATE (0.7 nM) at 30 min. The cell binding reached a plateau after 30 min of incubation with the radiopharmaceutical. The cell binding reduced to 5.5% ± 0.2% (25% inhibition) when incubated with 100 nM of cold DOTATATE, indicating the specificity of ⁹⁰Y-DOTATATE for SSTR-2 antigen. Nonspecific MCF7 cells showed only background counts. Figure 7A shows the total binding at different incubation times.

FIG. 7.

(A) In vitro cell-binding analysis with SSTR-2-expressing AR42J cells for 15, 30, 60, and 120 min. MCF7 Cells as negative control; (B) Cell Internalization assay in acidic buffer. In competition assay, cells were preincubated with cold DOTATATE (1 nM). Results are shown as average ± SD of triplicates. ***p < 0.001.

Percentage of the radiopharmaceuticals internalized into AR42J tumor cells at different incubation times is also shown in Figure 7B, which indicates the percentage of the total cell binding after acid treatment to remove the unbound fraction of radiopharmaceutical on the cell surface.

Biodistribution studies of ⁹⁰Y-DOTATATE in male nude mice

Biodistribution profiles of ⁹⁰Y-DOTATATE in male nude mice carrying AR42J pancreatic adenocarcinoma xenograft tumor are shown in Figure 8A. In the biodistribution study, the tumor uptake value (2.9% ± 0.8% dose/g) observed using ⁹⁰Y-DOTATATE was 14.6-fold higher compared with blood (0.2% ± 0.14% dose/g), which is statistically significant (p < 0.01).

FIG. 8.

(A) PET/CT ⁹⁰Y-DOTATATE scan (Injected 1mCi/mice, used Isoflurane anesthesia) of the nude mice carrying AR-42J Pancreatic Adenocarcinoma Xenograft tumor – 6 h postadministration; Coronal PET CT slice images of single mice; (B) Biodistribution Data of ⁹⁰Y-DOTATATE in SSTR-2-expressing tumor-bearing nude mice (injected 100 μCi/mice, used Isoflurane anesthesia); (C) Radio-TLC chromatogram of urine sample (24 h p.i) of male nude mice injected with ⁹⁰Y-DOTATATE. Color images are available online.

⁹⁰Y-DOTATATE was found to be cleared predominantly by the renal route. Radioactivity in the blood and most of the organs decreased after 48 h postinjection. High uptake and long-term retention of radioactivity were found in the kidney (9.96% ± 1.56% dose/g), which is in accordance with our scintigraphy data as shown in Figure 8B. Pharmacokinetic studies in male nude mice confirmed the in vivo stability of the product, as confirmed by the urine sample TLC 24 h postinjection (Fig. 8C).

Clinical efficacy of ⁹⁰Y-DOTATATE

The whole-body post-therapy Bremsstrahlung image (Fig. 9A) of patient with pancreatic NET involving liver metastasis after 1st cycle, whereas Bremsstrahlung image (Fig. 9B) of the same patient post 2nd cycle of ⁹⁰Y-DOTATATE therapy shows ideal localization of radiopharmaceutical in tumor. The pretherapy ⁶⁸Ga-DOTATATE image (Fig. 9C) was similar to post-therapy (after 1st cycle) Bremsstrahlung image of the same patient, showing significant tumor uptake of indigenously produced ⁹⁰Y-DOTATATE.

FIG. 9.

(A) Whole Body Anterior and Posterior Bremsstrahlung Images of the patient (post 1st therapy) showing good uptake of ⁹⁰Y-DOTATATE in the liver lesion; (B) Bremsstrahlung images of the same patient (after 2nd cycle) showing comparatively reduced uptake of ⁹⁰Y-DOTATATE in the same lesions seen above; (C) Whole-body PET/CT ⁶⁸Ga-DOTATATE scan of the patient before undergoing ⁹⁰Y-DOTATATE therapy; (D) ⁶⁸Ga-DOTATATE scan (after 3 months) of the same patient after undergoing 1st ⁹⁰Y-DOTATATE therapy with reduced uptake in the lesions as compared with the previous scan; (E) Regional PET/CT images of ⁹⁰Y-DOTATATE after the 1st and 2nd therapy. Color images are available online.

On follow-up after 3 months, post-therapy scan using ⁶⁸Ga-DOTATATE of the same patient showed reduction in the tumor uptake (Fig. 9D), which corroborates with excellent clinical efficacy of the administered ⁹⁰Y-DOTATATE. Post-therapy, PET/CT scan was carried out for the same patient after 1st and 2nd cycle of ⁹⁰Y-DOTATATE treatment (Fig. 9E) to study the imaging characteristics of ⁹⁰Y-DOTATATE with lower positron flux.

Clinical significance and response of patients who underwent ⁹⁰Y-DOTATATE PRRT

The PRRT therapy with⁹⁰Y-DOTATATE was initially adopted in our institute in Sequential duo-PRRT format²⁴; in this protocol, patients with tumor lesion size of >5 cm and no response to two cycles of ¹⁷⁷Lu-DOTATATE PRRT were considered for ⁹⁰Y-DOTATATE as part of sequential duo-PRRT with ¹⁷⁷Lu-DOTATATE. The ⁹⁰Y-DOTATATE therapy (in the sequential duo-PRRT regimen) was tolerated well by all 12 patients, with mild nausea in 2 patients (a standard observation in most PRRTs due to amino acid coinfusion) and transient grade I hematotoxicity in 2 patients reverting to normal between 10 and 12 weeks following ⁹⁰Y-DOTATATE treatment, with no nephrotoxicity or hepatotoxicity, until the follow-up period.

There was excellent tracer uptake in the known lesions on post-PRRT ⁹⁰Y-DOTATATE imaging (the Bremsstrahlung and PET/CT imaging were compared with previously done ⁶⁸Ga-DOTATATE PET/CT and ¹⁷⁷Lu-DOTATATE post-therapy scans). A clinical patient example is depicted in Figure 9A–D in the Clinical efficacy of 90Y-DOTATATE section.

Discussion

β⁻ radionuclide impurity

The presence of various actinides (²³⁸U, ²³⁹Pu, ²⁴¹Am, ²⁴⁴Cm) and fission products (¹³⁷Cs, ¹⁴⁴Ce, ¹⁰⁶Ru, ¹⁴⁷Pm, ¹²⁵Sb, ¹⁰⁶Rh, ⁹⁰Sr¹²⁹I, ¹⁰⁶Pd, ^152/154Eu) makes the HLLW solutions radiotoxic and hazardous.²⁵ Separation of purified ⁹⁰Sr therefore poses a challenging task. The ⁹⁰Sr (T_1/2: 28 years) attains secular equilibrium with ⁹⁰Y (T_1/2: 2.67 d) in a short period and constitutes to be a perpetual source of ⁹⁰Y. Some of these actinides and fission products contribute for α, β⁻, and γ-emitting radionuclidic impurities in ⁹⁰Y-Acetate solution. The major source of β⁻-emitting radionuclide impurity in ⁹⁰Y-Acetate solution is ⁹⁰Sr. The specified levels of ⁹⁰Sr content in clinical-grade ⁹⁰Y-Acetate solution should be less than ≤2.5 μCi/Ci of ⁹⁰Y. Therefore, it becomes important to accurately determine such low content of ⁹⁰Sr. The estimation of ⁹⁰Sr content by EPC technique was not deemed suitable for estimating the ⁹⁰Sr content in the ⁹⁰Y-Acetate solution. A second method, which is essentially carrying out β⁻ spectrometry enables the accurate estimation of the levels of ⁹⁰Sr in ⁹⁰Y-Acetate solution.

Gross γ radionuclide impurity

The contributing γ-emitting radionuclidic impurities in ⁹⁰Y-Acetate solution are ¹⁴⁴Ce and ¹²⁵Sb. The recording of γ spectrum in 64 k (2 h) and 4 k (24 h) channels allow the determination of high-energy, low-abundance γ-emitting radionuclidic impurities (if any) in the presence of long-lived γ-emitting radionuclides in the ⁹⁰Y-Acetate solution.

Gross α radionuclide impurity

The determination of the limit of α radionuclide in ⁹⁰Y-Acetate solution is critical and poses a major challenge. This is particularly difficult since no pharmacopeia reference is available stating the acceptable limits of gross α activity. Hence for establishing the limit, we had to focus on assessing the content of the radionuclides, which contribute to the α impurities in ⁹⁰Y-Acetate solution. Considering the extraction of ⁹⁰Y from HLLW, the major α radionuclides, which could be possibly present are ²⁴¹Am or ²³⁹Pu. The hazard factor (HF) for ²⁴¹Am (3.85 × 10⁹) is 80–90 times more compared with ²³⁹Pu (1.91 × 10⁷).²⁵ Hence the gross α impurities in ⁹⁰Y-Acetate solution are attributable to the presence of ²⁴¹Am.

Taking the annual limit of intake for ²⁴¹Am (Ingestion limit: 30,000 Bq or 810 nCi, Inhalation limit: 220 Bq or 5.94 nCi) into consideration,²⁶ the permissible limits of gross α impurity levels were fixed at ≤1.0 nCi/Ci of ⁹⁰Y. The single patient therapeutic PRRT dose does not exceed 120 mCi and the patient undergoes three cycles in a year. Considering the presence of 1 nCi of gross α levels in ⁹⁰Y-Acetate solution, the patient will receive 0.36 nCi of gross α in a single year, which is much less than the permissible limit. For detecting such low levels of gross α in ⁹⁰Y-Acetate solutions, the commonly used liquid scintillation-based counting will not suffice, hence ZnS (Ag)-based scintillation counting and solid-state track detector-based scanning should be adopted.

RCP of ⁹⁰Y-Acetate

The presence of a single chemical species of ⁹⁰Y is a very important factor for radiolabeling with peptides or monoclonal antibody. There is a finite chance that ⁹⁰Y(NO₃)₃ be present apart from the desired radiochemical⁹⁰Y-Acetate. During the transportation of ⁹⁰Y from the feed chamber (containing 4 M HNO₃) of second-stage SLM generator to receiver chamber (containing 1 M CH₃COOH) through CMPO-impregnated PTFE membrane, there is a possibility of existing ⁹⁰Y(NO₃)₃ in final ⁹⁰Y-Acetate solution. The NO₃ ⁻ concentration has been estimated and the presence of ⁹⁰Y(NO₃)₃ has been ruled out.

Nitrate ion concentration in ⁹⁰Y-Acetate

Stripping of nitrate ions from 4 M HNO₃ present in the feed chamber of second stage SLM generator to receiver compartment (containing 1 M CH₃COOH) through CMPO-impregnated PTFE membrane increases the nitrate ion concentration in ⁹⁰Y-Acetate solution. Higher levels of nitrate content in any pharmaceutical and food product are toxic to human beings. The average daily intake of nitrate ion is 7.0 mg/kg of body weight per day, whereas LD₅₀ values of sodium nitrate in rabbit is 1600–9000 mg per kg of body weight.²² However, the level of nitrate ion concentration in the ⁹⁰Y-Acetate solution was found to be 1.25 mg/kg of body weight in a single cycle of PRRT (single cycle dose is 120 mCi). Considering a patient undergoes three cycles of PRRT in a year, 3.75 mg/kg of body weight will be the total intake of nitrate ions in patients undergoing PRRT in a whole year.

Metal contents in ⁹⁰Y-Acetate

The presence of a competing metal ion M³⁺, such as Fe³⁺, Cu²⁺, Zn²⁺ etc. drastically lowers the radiolabeling yield of the resultant ⁹⁰Y-labeled peptides. The competing M³⁺ metal ions arise in ⁹⁰Y-Acetate solutions during separation of ⁹⁰Sr from Purex high-level waste. These transition metals have high thermodynamic stability with chelators used in radiolabeling reactions. Such metals are usually present in the purified ⁹⁰Sr(NO₃)₂ feed solution at comparatively higher concentrations than permissible limits. However, they do not get transported through KSM-17-impregnated PTFE membrane to the receiver chamber of first-stage SLM generator containing 4 M HNO₃. The levels of intervening metal content in ⁹⁰Y-Acetate solution could be substantially reduced by using ultrapure grade 4 M HNO₃ and 1 M CH₃COOH.

Phosphorus-based impurities in ⁹⁰Y-Acetate

The phosphorous content in the ⁹⁰Y-Acetate solution may increase, due to radiolytic degradation of the KSM-17 and CMPO (both are phosphorus-based polymers)-impregnated PTFE membrane during the process of separation, even though these PTFE membranes are meant for single use. The absence of any peak in ³¹P-proton-decoupled NMR and comparison with that of the ³¹P-proton-decoupled NMR of CMPO and KSM-17 (which individually shows a single peak) establishes that the ⁹⁰Y-Acetate solution is devoid of any phosphorous content.

Stable ⁹⁰Zr content in ⁹⁰Y-Acetate

There is no defined limit of stable ⁹⁰Zr in ⁹⁰Y-Acetate solution available in the reported literature. However, at the same time, the stable ⁹⁰Zr is toxic to humans. ⁹⁰Zr obtained from standard daily diet is 3.5 mg, which means that the allowed daily intake is 3.5 mg, per individual. This corresponds to 1280 mg of ⁹⁰Zr for a 70 kg body weight individual in a year from standard dietary sources.²³ Eventually, ⁹⁰Y decays to ⁹⁰Zr, hence maximum ⁹⁰Zr produced by decay of repeated doses from ⁹⁰Y is 1 μg in a single year.²³ Apart from decay of ⁹⁰Y to ⁹⁰Zr, there are also chances for increased levels of ⁹⁰Zr in ⁹⁰Y-Acetate solution during two-stage SLM-based separation process and various purification processes of HLLW.

The level of ⁹⁰Zr in our indigenously sourced ⁹⁰Y-Acetate solution was 0.4 ng/kg of body weight in a single cycle of PRRT (single cycle dose is 100 mCi). Considering a patient undergoes three cycles of PRRT in a year, 1.2 ng/kg of body weight will be the total intake of ⁹⁰Zr in patients in the whole year. The intake of stable ⁹⁰Zr in patients undergoing PRRT will be <90 ng/70 kg body weight in any circumstances, which is much less than the allowed annual intake of ⁹⁰Zr.

Radiolabeling of DOTATATE using indigenously sourced ⁹⁰Y-Acetate

With respect to the radiolabeling of peptide with ⁹⁰Y, the ⁹⁰Y-Acetate has an advantage over that of ⁹⁰Y-Chloride. Generally radiolabeling is carried out in acetate buffer either in the form of sodium acetate or ammonium acetate. Therefore, by using ⁹⁰Y-Acetate, the pH of the reaction mixture can be adjusted to 4.0 with less volume of buffer without addition of any NaOH.

Radiolabeling was carried out using both the buffers that is, sodium acetate and ammonium acetate under same reaction conditions. However, 0.2 M ammonium acetate buffer has an advantage over that of 0.2 M sodium acetate buffer, since the limited volume of 0.2 M CH₃COONH₄ buffer (3 mL) has a good buffering capacity to raise the pH of reaction mixture from 2.0 to 4.0 for 100–120 mCi of ⁹⁰Y-Acetate. Comparatively CH₃COONH₄ buffer stabilizes the radiolabeling reaction without any 2, 5-dihydroxybenzoic acid or ascorbic acid.

Incomplete radiolabeling of ⁹⁰Y-DOTATATE was observed when 2, 5-dihydroxybenzoic acid was added during the radiolabeling process. Therefore, a C18 Sep-Pak purification postradiolabeling becomes mandatory to obtain the desired RCP of ⁹⁰Y-DOTATATE (>98%). The addition of a required concentration of stabilizer (2, 5-dihydroxybenzoic acid) after radiolabeling process allows to stabilize ⁹⁰Y-DOTATATE with RCP >98% without any C18 Sep-Pak purification. The addition of stabilizer postradiolabeling process also increases the in vitro stability of ⁹⁰Y-DOTATATE up to 72 h on storage at −20°C with retention of RCP >98%.

Quality control of ⁹⁰Y-DOTATATE

Apart from Radio-TLC, Radio-HPLC allows to detect the presence of radiochemical impurities (if any) at low levels in the ⁹⁰Y-DOTATATE.

The choice of gradient mode, namely steep gradient or shallow gradient, is an important step toward determining of RCP in ⁹⁰Y-DOTATATE. The chances of detecting low levels of radiochemical impurities (if any), in ⁹⁰Y-DOTATATE, using steep gradients, is by far the least as compared with the results when a shallow gradient mode is used. Hence the shallow gradient mode HPLC must be adopted for determining RCP of ⁹⁰Y-DOTATATE.

In vitro and serum stability of ⁹⁰Y-DOTATATE

Different storage conditions of ⁹⁰Y-DOTATATE were evaluated to optimize the in vitro stability of the product. ⁹⁰Y-DOTATATE started degrading at 24 and 6 h postradiolabeling on storage at 4°C with stabilizer and in both cases RCP falls <90%. Therefore, the recommended use of ⁹⁰Y-DOTATATE is within 3 h postformulation upon storage at room temperature and 4°C.

In vitro and in vivo studies of ⁹⁰Y-DOTATATE

In vitro cell-binding studies carried out in AR42J cells showed high specificity of ⁹⁰Y-DOTATATE for SSTR-2. In the in vitro experiments, the cell-binding percentage of 30% could be obtained with the ⁹⁰Y-DOTATATE and the results were comparable with values reported earlier. In vitro cell internalization and competition binding assays ascertain the specificity and efficacy of the radiopharmaceutical.

Detailed bioevaluation studies in AR42J xenograft tumor-bearing nude mice further confirmed the specificity of the product. High uptake in the tumor target and long retention thereof ascertains the effective dose delivery to the NET cancer tissue. Prolonged retention of the ⁹⁰Y-DOTATATE in the kidney as is known confirms the renal mode of excretion. As reported earlier, the damage incurred to kidney can be minimized by renal protection through preadministration of positively charged amino acid.

Therapeutic value of ⁹⁰Y-DOTATATE

Post ⁹⁰Y-DOTATATE therapy, Bremsstrahlung image of the same NET patient, using γ camera corroborated with the diagnostic ⁶⁸Ga-DOTATATE scan, thereby proving efficient radiolabeling and excellent tumor uptake with ideal target to background contrast.

Post 1st cycle (3 months) of ⁹⁰Y-DOTATATE therapy, ⁶⁸Ga-DOTATATE scan of the same patient showed reduced uptake of the radiopharmaceutical in the liver, thereby proving excellent therapeutic efficacy of the administered ⁹⁰Y-DOTATATE. Post 1st cycle (3 months) of ⁹⁰Y-DOTATATE therapy, ⁶⁸Ga-DOTATATE scan of the same patient showed reduced uptake of the radiopharmaceutical in the liver, which was indicative of the possible therapeutic efficacy of the administered ⁹⁰Y-DOTATATE.

Since the sequential duo PRRT with ⁹⁰Y-DOTATATE was not completed in this group of patients, definitive data on therapeutic response assessment especially on scan/imaging response of the large-sized tumors, was not possible at this stage. However, the PRRT procedure was indicative of the tolerability of the agent with no observation of side effects.

Conclusions

Using a SLM-based ⁹⁰Sr/⁹⁰Y generator, where the feed ⁹⁰Sr is obtained from HLLW, clinical-grade ⁹⁰Y-Acetate could be obtained. This radiochemical precursor was successfully used in formulation of therapeutic doses of ⁹⁰Y-DOTATATE. The quality control parameters of ⁹⁰Y-Acetate were validated and compared with that of the pharmacopeia standard. This ⁹⁰Y-Acetate has been utilized in the formulation of patient doses of ⁹⁰Y-DOTATATE.

To the best of the authors' knowledge, the monograph of neither ⁹⁰Y-Acetate nor ⁹⁰Y-DOTATATE has been included in any pharmacopeia. The detailed validation of the quality control method of ⁹⁰Y-Acetate was necessary for obtaining regulatory clearance of the radiochemical precursor. Successful formulation of ⁹⁰Y-DOTATATE and its clinical translation exemplified recovery of “wealth from waste”. Clinical results in patients showed expected distribution in patients with large volume NET (≥ 5 cm) and its metastases.

Footnotes

Acknowledgments

The authors gratefully acknowledge the support and encouragement received from Dr K. Agarwal, erstwhile Director, Nuclear Recycle Group, BARC, Mumbai, India, and Dr C. P. Kaushik, Associate Director, Nuclear Recycle Group, BARC, Mumbai, India.

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for the work.

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On the Separation of Yttrium-90 from High-Level Liquid Waste: Purification to Clinical-Grade Radiochemical Precursor,Clinical Translation in Formulation of 90 Y-DOTATATE Patient Dose

Abstract

Introduction:

Methods:

Results:

Conclusions:

Introduction

Materials and Methods

Separation and purification of 90Sr from HLLW

Additional purification of 90Sr

Separation of clinical-grade 90Y in 90Y-Acetate form using the two-stage SLM generator

Physicochemical and biological quality control of 90Y-Acetate

Radiolabeling of DOTATATE with 90Y-Acetate and its quality control

In vitro studies of 90Y-DOTATATE

Cell binding

Cell internalization

Competition assay

In vivo biodistribution studies of 90Y-DOTATATE

Clinical studies of 90Y-DOTATATE

Results

RNP of 90Y-Acetate

The RCP of 90Y-Acetate

Metallic impurities in 90Y-Acetate

Nitrate ions, phosphorus, and stable 90Zr contents in 90Y-Acetate

EL and sterility of 90Y-Acetate