Automated Module Radiolabeling of Peptides and Antibodies with Gallium-68,Lutetium-177 and Iodine-131

Introduction

The continuing rapid development of therapeutic nuclear oncology with the advent of novel radioimmunotherapy and radiopeptide treatment of metastatic cancer is dependent upon practical, affordable, and safe provision of therapeutic activities of targeted radiopharmaceuticals.

Safety not only requires good manufacturing practice and standard operating procedures to ensure sterility and purity of the radiopharmaceutical, but also incorporates radiation protection of the radiopharmaceutical chemist during manufacture, quality control, and dispensing.

Typical activities of radiopharmaceuticals prepared for therapeutic applications are in the range 10–50 GBq, which require special remote shielded manipulation to avoid radiation exposure, particularly to fingers of the radiopharmacist. The radiolabeling of monoclonal antibody proteins and peptides for targeted tumor therapy with high activities in relatively small volumes inevitably causes radiolysis which is minimized by in-house preparation of each radiopharmaceutical immediately before administration to the patient. Such customized on-site radiolabeling is cost-effective and reliable, given that delivery of the therapy radionuclides Iodine-131 and Lutetium-177 from a reactor is elective and PET diagnostic Gallium-68 is available daily from an in-house Germanium-68 generator.

In our 10 year experience of ¹³¹I-rituximab radioimmunotherapy of non-Hodgkin lymphoma ¹ and ¹⁷⁷Lu-octreotate radiopeptide therapy of neuroendocrine tumors ² we have progressed from manual radiolabeling ³ through semiautomated methodology to the current fully automated synthesis achieved by reprogramming a commercially available module as described in this paper. This methodology is now also routinely applied in our hospital to synthesize ⁶⁸Ga peptides for PET diagnostic imaging.

Materials and Methods

⁶⁸Ga-DOTA-Tyr³-octreotate

Gallium-68 was obtained from a tin-dioxide-based ⁶⁸Ge/⁶⁸Ga generator (IDB) as ⁶⁸GaCl₄, ⁶⁸Ga (T _1/2= 68 minutes) being continuously produced by decay of its radioactive parent ⁶⁸Ge (T _1/2= 270.8 days). Elution of the generator was performed in a shielded workstation with 0.6 M HCl Suprapure (Sigma—Aldrich). A Teflon tube was inserted into the eluate vial for transfer of the ⁶⁸GaCl₄ to the reaction vial within the Synthera^® module.

Radiolabeling was remotely performed within the shielded, sterile module according to the general method described by Breeman et al. ⁴ Briefly, sodium acetate 2.5 M was used to adjust the pH of the ⁶⁸GaCl₄ eluate to 3.0–4.0. DOTA- Tyr³- octreotate, synthesized and supplied as a lyophilized powder in sterile vials by Auspep, was then added as a 20–30 nmol solution. After heating for 10 minutes at 98°C, 10 μL 4 mM EDTA (Sigma—Aldrich) was added (Fig. 1).

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FIG. 1.

⁶⁸Ga-DOTA-Tyr³-octreotate flowchart of the Synthera^® module showing vials 1 (sodium acetate), 2 (DOTA-octreotate), and 3 (EDTA) mounted on the disposable, sterile cassette.

The radiolabeled product was transferred to a sterile vial after end filtration using a 0.2 μm filter (Acrodisc 0.2 μm; Pall).Radiochemical yield and purity was determined by ITLC-SG in a sodium citrate buffer 0.1 M pH 5.5.

HPLC quality control analysis was performed (Shimadzu). A reversed phase column C₁₈, 5 μm, 4.6×250 mm (Grace) was used as stationary phase with a solvent system of 0.1% trifluoroacetic acid (TFA) in H₂O (solvent A) and 0.1% TFA in acetonitrile (solvent B) in a gradient protocol; 0–10 minutes 18%–60% solvent B, 10–12 minutes 60% solvent B. The flow-rate was 0.6 mL/min.

¹⁷⁷Lu-DOTA-Tyr³-octreotate

Lutetium-177 trichloride was supplied by IDB Holland. Radiolabeling of DOTA-Tyr³-octreotate was performed by adding 400 μg peptide to the ¹⁷⁷LuCl₃ (10–50 GBq), followed by heating for 30 minutes at 98°C. After heating l ml EDTA (4 mg/mL) was added and the solution was transferred to a sterile vial after end filtration using a 0.22 μm filter (Fig. 2). Radiochemical yield and purity was performed by ITLC-SG in a sodium citrate buffer 0.1 M pH 5.5.

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FIG. 2.

¹⁷⁷Lu-DOTA-Tyr³-octreotate flowchart of the Synthera^® module showing vials 2 (DOTA-octreotate) and 3 (DTPA) mounted on the disposable, sterile cassette.

HPLC quality control analysis was performed (Shimadzu). A reversed phase C₁₈ column 5 μm, 4.6×250 mm (Grace) was used as stationary phase with a solvent system of 0.1% TFA in H₂O (solvent A) and 0.1% TFA in acetonitrile (solvent B) in a gradient protocol; 0–10 minutes 100% solvent A, 10–40 minutes a linear gradient from 5% B to 90% B. The flow-rate was 0.6 mL/min.

¹³¹I-rituximab

Unstabilized sodium ¹³¹I iodide was supplied by Ansto Health.

Radiolabeling of rituximab was performed using a chloramine-T method. ³ Purification of the radiolabeled product was done on a Pd-10 column (Sephadec G-25; GE Healthcare). The radiolabeled product was transferred to a sterile vial after end filtration using a 0.22 μm filter (Fig. 3).

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FIG. 3.

¹³¹I-rituxmab flowchart of the Synthera^® module showing vials 2 (rituximab+chloramine-T), 3 (sodium thiosulfate), and 4 (PBS) and Pd-10 column mounted on the disposable, sterile cassette.

Radiochemical yield and purity was performed by ITLC-SG in 85% methanol.

HPLC analysis was performed on a Shimadzu HPLC system. A macrosphere column (7 μm, 7.5×300 mm; Alltech) was used as stationary phase with sodium phosphate 0.05 M pH 6.75 as a solvent. The flow-rate was 1.0 mL/min.

Results

Discussion

The practice of therapeutic nuclear medicine has undergone a renaissance with the advent of novel tumor-targeted radiopharmaceuticals which may be manufactured in hospital radiopharmacies for customized radionuclide therapy of cancer patients. The efficacy and safety of such innovative personalized approaches to the control of disseminated cancer, such as radiopeptide therapy of neuroendocrine tumor with ¹⁷⁷Lu-octreotate, has been demonstrated over the past 10 years. ⁵ However, such advances in radionuclide therapy have been often made without formal development of approved agents by pharma industry and in the case of radiopeptide therapy, no randomized controlled clinical trials have been performed. ⁶

Clinical evaluation of radiopeptide therapy of neuroendocrine tumors and other cancers thus depends upon multicenter physician-sponsored studies, performed within funding and regulatory agency constraints. Thus the ability to prepare affordable, safe, effective therapeutic radiopharmaceuticals on-site in existing radiopharmacies in hospital Nuclear Medicine departments is a prerequisite for the clinical application of novel targeted nuclear medicine therapy. ⁷

Many Nuclear Medicine departments offer a PET diagnostic service based upon ¹⁸ F-FDG prepared in a commercially available synthetic module, of which the IBA Synthera^® is but one example. We have reprogrammed the basic Synthera^® module simply by writing an individual script file for automated synthesis of each desired therapeutic radiopharmaceutical and PET diagnostic agent, and have accordingly adapted the standard single use sterile cassette system.

Manual handling of high amounts of radioactivity of ⁶⁸GaCl₃ results in a high finger-dose exposure. For routine production of ⁶⁸Ga-DOTA-Tyr³-octreotate, the automated method resulted in a significant decrease of radiation absorbed dose to the operator. Further, an increase of the overall yield was observed, due to shorter synthesis time. The use of a cassette system makes it also possible to make adjustments to the synthesis, such as buffers, ⁸ in line prepurification of ⁶⁸GaCl₃ ^9,10 type of chelator ^11,12 or peptide ^13,14 to optimize yields and purity.

The amount of DOTA-Tyr³-octreotate required for 100% incorporation of lutetium-177 into the peptide increases with time from production of ¹⁷⁷LuCl₃. ⁷ Given the relatively long transit time of ¹⁷⁷LuCl₃ to Australia from Holland, we used a higher fixed amount of 400 μg than the standard 250 μg used for the same peptide in Europe. Clinical results of the lower specific activity ¹⁷⁷Lu-octreotate are however comparable. ^2,5

Mainstream clinical use of anti-CD20 monoclonal antibody radioimmunotherapy of non-Hodgkin lymphoma has been constrained by perceived lack of affordability and practicality. ^15,16 However, the imminent availability of generic anti CD20 Mabs is expected to significantly lower costs and a safe, practical automated radiolabeling system for ¹³¹I-rituximab will provide widespread access to radioimmunotherapy of non-Hodgkin lymphoma throughout developing countries. Similarly the same module may be programmed for synthesis of ¹⁷⁷Lu-rituximab ^17,18 where local reactor-produced ¹⁷⁷LuCl₃ may be more cost-effective.

Ready availability of high-yield, radiochemically pure, clinical grade tumor-targeted therapeutic radiopharmaceuticals in-house facilitates research and development of personalized radionuclide treatment of cancer. Serial labeling of several radiopharmaceuticals on the same day, using the fully automated synthetic module does not expose the manufacturing radiopharmaceutical chemist to significant radiation exposure, which was a limiting factor when therapeutic radiopharmaceuticals were manually prepared.

Conclusions

The automated modular synthesis of therapeutic and PET diagnostic tumor-targeted agents is reliable, rapid and remotely controlled to achieve reproducible, quality-controlled, high-yield radiopharmaceuticals without radiation exposure to the laboratory staff and may be recommended as the standard operating procedure for preparation of radiopharmaceuticals for nuclear oncology.