Validation of a radiosynthesis method and a novel quality control system for [68 Ga]Ga-MAA: is TLC enough to assess radiopharmaceutical quality?

Abstract

Background: Technetium-99 m-labelled macroaggregated human serum albumin ([99mTc]Tc-MAA) is commonly used for lung perfusion scintigraphy. The European Pharmacopoeia (Eu.Ph.) specifies thin-layer chromatography (TLC) as the only method to assess its radiochemical purity (RCP). Similarly, TLC is the sole method reported in the literature to evaluate the RCP of Gallium-68-labelled MAA [⁶⁸ Ga]Ga-MAA, recently introduced for lung perfusion PET/CT imaging. Since [⁶⁸ Ga]Ga-MAA is prepared from commercial kits originally designed for the preparation of [99mTc]Tc-MAA, it is essential to optimize and validate the preparation methods for [⁶⁸ Ga]Ga-MAA.

Results: We tested a novel, simplified method for the preparation of [⁶⁸ Ga]Ga-MAA that does not require organic solvents, prewash or final purification steps to remove radioactive impurities. We assessed the final product using radio-TLC, radio-UV-HPLC, and radio SDS-PAGE. Overall, our quality control (QC) method successfully detected [⁶⁸ Ga]Ga-MAA along with all potential impurities, including free Ga-68, [⁶⁸ Ga]Ga-HSA, unlabeled HSA, which may occur during labelling process and HEPES residual, a non-toxic but non-human-approved contaminant, used as buffer solution. We then applied our QC system to [⁶⁸ Ga]Ga-MAA prepared under different conditions (25°-40°-75°-95 °C), thus defining the optimal temperature for labelling. Scanning Electron Microscopy (SEM) analysis of the products obtained through our novel method confirmed that most [⁶⁸ Ga]Ga-MAA particles preserved the morphological structure and size distribution of unlabeled MAA, with a particle diameter range of 25-50 μm, assuring diagnostic efficacy.

Conclusions: We optimized a novel method to prepare [⁶⁸ Ga]Ga-MAA through a QC system capable of monitoring all impurities of the final products.

Keywords: High pressure liquid chromatography; Lung perfusion imaging; Morphological structure and size distribution; Radiopharmaceuticals; SDS-PAGE; [68 Ga]Ga-macroaggregated human serum albumin.

Fig. 1

Radio-TLC of a [⁶⁸ Ga]GaCl₃, b [⁶⁸ Ga]-HSA, and c [⁶⁸ Ga]Ga-MAA crude product, obtained with a radiolabelling temperature of 95 °C

Fig. 2

Radio-UV-HPLC chromatograms of [⁶⁸ Ga]Ga-MAA obtained at different temperatures: a 25 °C, b 45 °C, c 75 °C, d 95 °C

Fig. 3

Calibration curve obtained with a the values of peak areas of five different concentrations (20, 10, 5, 3, 2, 1 mg/mL) of HSA; b the average values of peak areas of [⁶⁸ Ga]Ga-HSA

Fig. 4

a 12% SDS-PAGE of resuspended Pulmocis® (total), and the retentate and permeate upon filtration. SDS-PAGE samples were prepared at 100 °C for 20 min in the presence of 2 mM 2-mercaptoethanol (condition a), at 100 °C for 45 min in the presence of 2 mM 2-mercaptoethanol (condition b) and at 37 °C for 20 min with no 2-mercaptoethanol (condition c). The three original samples were diluted to achieve similar protein concentration for densitometric analysis; b radio SDS-PAGE of [⁶⁸ Ga]Ga-MAA Pulmocis® separated by filtration

Fig. 5

Microscope images of Pulmocis® sample after reconstitution. a Magnification 100x b Magnification 200x

Fig. 6

Microscope images of [⁶⁸ Ga]Ga-Pulmocis sample a 100 × and b 200 x) after the labelling procedure

Fig. 7

Particle size distribution analysis reported as cumulative undersize volume diameter distribution (red line) or percentage of frequency of particles by volume diameter (blue histogram) of Pulmocis® a and [⁶⁸ Ga]Ga-MAA b

Fig. 8

Synthesis production scheme of [⁶⁸ Ga]Ga-MAA: 1. 68Ge/68 Ga generator elution and [⁶⁸ Ga]GaCl₃ eluate purification. 2. Mixing the [⁶⁸ Ga]GaCl₃ eluate with the MAA. 3. Heat the reaction solution at (25°–40°–75°–95 °C). 3. Filtration of [⁶⁸ Ga]Ga-MAA. 4. Collection of [⁶⁸ Ga]Ga-MAA for the final formulation