Development and Validation of an Analytical HPLC Method to Assess Chemical and Radiochemical Purity of [(68)Ga]Ga-NODAGA-Exendin-4 Produced by a Fully Automated Method

Background: Glucagon-like peptide 1 receptor (GLP-1R) is preferentially expressed in pancreatic islets, especially in β-cells, and highly expressed in human insulinomas and gastrinomas. In recent years several GLP-1R–avid radioligands have been developed to image insulin-secreting tumors or to provide a tentative quantitative in vivo biomarker of pancreatic β-cell mass. Exendin-4, a 39-amino acid peptide with high binding affinity to GLP-1R, has been labeled with Ga-68 for imaging with positron emission tomography (PET). Preparation conditions may influence the quality and in vivo behavior of tracers. Starting from a published synthesis and quality controls (QCs) procedure, we have developed and validated a new rapid and simple UV-Radio-HPLC method to test the chemical and radiochemical purity of [(68)Ga]Ga-NODAGA-exendin-4, to be used in the clinical routine. Methods: Ga-68 was obtained from a (68)Ge/(68)Ga Generator (GalliaPharma(®)) and purified using a cationic-exchange cartridge on an automated synthesis module (Scintomics GRP(®)). NODAGA-exendin-4 contained in the reactor (10 µg) was reconstituted with HEPES and ascorbic acid. The reaction mixture was incubated at 100 °C. The product was purified through HLB cartridge, diluted, and sterilized. To validate the proposed UV-Radio-HPLC method, a stepwise approach was used, as defined in the guidance document released by the International Conference on Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH), adopted by the European Medicines Agency (CMP/ICH/381/95 2014). The assessed parameters are specificity, linearity, precision (repeatability), accuracy, and limit of quantification. Therefore, a range of concentrations of Ga-NODAGA-exendin-4, NODAGA-exendin-4 (5, 4, 3.125, 1.25, 1, and 0.75 μg/mL) and [(68)Ga]Ga-NODAGA-exendin-4 were analyzed. To validate the entire production process, three consecutive batches of [(68)Ga]Ga-NODAGA-exendin-4 were tested. Results: Excellent linearity was found between 5–0.75 μg/mL for both the analytes (NODAGA-exendin-4 and (68)Ga-NODAGA-exendin-4), with a correlation coefficient (R(2)) for calibration curves equal to 0.999, average coefficients of variation (CV%) < 2% (0.45% and 0.39%) and average per cent deviation value of bias from 100%, of 0.06% and 0.04%, respectively. The calibration curve for the determination of [(68)Ga]Ga-NODAGA-exendin-4 was linear with a R(2) of 0.993 and CV% < 2% (1.97%), in accordance to acceptance criteria. The intra-day and inter-day precision of our method was statistically confirmed using 10 μg of peptide. The mean radiochemical yield was 45 ± 2.4% in all the three validation batches of [(68)Ga]Ga-NODAGA-exendin-4. The radiochemical purity of [(68)Ga]Ga-NODAGA-exendin-4 was >95% (97.05%, 95.75% and 96.15%) in all the three batches. Conclusions: The developed UV-Radio-HPLC method to assess the radiochemical and chemical purity of [(68)Ga]Ga-NODAGA-exendin-4 is rapid, accurate and reproducible like its fully automated production. It allows the routine use of this PET tracer as a diagnostic tool for PET imaging of GLP-1R expression in vivo, ensuring patient safety.