[18F]ASIS Production under Good Manufacturing Practices (GMP)

Objectives Tissue factor (TF) is a transmembrane glycoprotein. The primary function of TF is activation of the clotting cascade but TF is also implicated in cancer progression, angiogenesis and metastasis via TF/FVIIa (activated Factor VII) -mediated activation of PARs (protease-activated receptors) [1]. TF/FVIIa-dependent activation increases tumor growth, and TF is therefore a potential target for assessment of cancer aggressiveness [1]. Active Site Inhibited Factor VIIa (ASIS) exhibits a five times higher affinity towards TF than Factor VIIa itself and is without pro-coagulant activity [3]. We have recently labeled Active Site Inhibited Factor VII (ASIS) with [18F]SFB which in in vivo mouse models shows promising results [2,4]. The aim of this work was to develop and produce [18F]ASIS according to GMP in preparation for clinical trials in human.

Methods All critical raw materials used in production of [18F]ASIS were of high quality (purity 蠅 98 %) and where relevant in compliance with European Pharmacopoeia monographs. All buffer solutions were autoclaved. FVIIa was provided by Novo Nordisk A/S in pharmaceutical grade. ASIS was produced by blocking the active site of FVIIa with Phe-Phe-Arg-chloromethyl ketone (FFR-cmk) and excess inhibitor removed by dialysis [3]. The 18F-containing prosthetic group, [18F]SFB, was produced in a three-step, onepot synthesis on a qualified TracerLab MXFDG module with SPE purification. Fractions of [18F]SFB in CH3CN were evaporated to dryness in a single vial and the conjugation reaction was performed by addition of a solution of ASIS (500 µl, 2.4-4.6 mg/mL) in either 50 mM HEPES (150 mM NaCl, 10 mM CaCl2) pH 7.4 or 0.1 M borate buffer (150 mM NaCl, 10 mM CaCl2) pH 8.5 and reaction at room temperature for 30 min. Subsequently, the final product was purified on a PD-10 desalting column and sterile filtered in a laminar airflow bench. [18F]ASIS was analyzed by HPLC and protein precipitation, and an intact TF binding was evaluated in pull-down experiments. Analytical methods were validated according to ICH Harmonised Tripartite Guideline: Validation of Analytical Methods: Methodology, ICH Topic Q2B guideline.

Results 4620 - 7741 MBq [18F]SFB was prepared in 22.5 ± 1,9% radiochemical yield (n=7) on TracerLab MXFDG. After evaporation coupling of [18F]SFB to ASIS was achieved in 12.2 ± 4.6% radiochemical yield (n=6). The radiochemical yield was affected by the evaporation procedure and optimal when small fractions (1 ml) of [18F]SFB were evaporated sequentially. 455 +/- 41 MBq [18F]ASIS was produced within 150 min with a radiochemical purity 蠅98% and specific radioactivity of 423 ± 4 MBq/mg. The product was stable in solution for 6 hours at room temperature. All test results were within the specification. The protein- associated activity of the labeled product was determined by protein precipitation to be >96% (n=2). Binding to soluble TF and the anti-FVIIa monoclonal antibody F1A2 was also confirmed.

Conclusions A procedure for GMP manufacturing of [18F]ASIS was developed. During preparation for GMP manufacturing of [18F]ASIS two different buffer systems were investigated to achieve optimal radiochemical yield and the highest TF binding. [18F]ASIS produced following GMP will be evaluated in vivo as a non-invasive agent for imaging of the aggressiveness of cancer in dogs and eventually in humans.