Sviluppo di un metodo per purificare il Reovirus 3 per studi di Viral Clearance, in accordo alle linee guide ICH Q5A (R1)

Viral Clearance Studies are part of biopharmaceutical manufacturing process evaluations and are designed to check the manufacturing process and to evaluate if the process itself is safe in case of viral contamination. The aim of a Viral Clearance Study is to assess the purification process capability to remove or inactivate viruses that may potentially contaminate the raw materials and viruses that could be introduced during manufacturing process. Viral Clearance Studies are designed and conducted to demonstrate the Viral Safety of biotechnology products, according to the International Guideline Q5A(R1) “Viral safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin” defined by International Conference on Harmonization (ICH) (Europe - USA - Japan). To perform a Viral Clearance Study, high titre and purified virus stocks are needed. A virus with a defined titre is added (“spiking”) to intermediate samples, depending on the analysed process steps. Subsequently, the material (samples + virus) is subjected to the purification step that needs to be evaluated, to assess virus inactivation and/or removal grades of the step. Spiked samples go through the downscaled purification step and the residual viral concentration is checked with in vitro tests to calculate the reduction factor. The most common methodologies to virus inactivation or removal in biopharmaceutical manufacturing processes are: pH Treatment, Solvent/Detergent, Heat treatment, Filtration and Chromatography. Viruses used for spiking are obtained by propagation in a specific cell line. In fact, each virus has an elective cellular substrate for its propagation and/or titration. In order to obtain a high titre and purified virus stocks, without losing their effectiveness, it is necessary to proceed with a series of steps that defines the virus purification process. Among them ultracentrifugation and filtration are the most significant. The following thesis, written during the internship period at the Istituto di Ricerche Biomediche "A. Marxer" (R.B.M.) – Merck Serono S.p.A. (Colleretto Giacosa, TO), analyses the key points of Reovirus 3 (REO3) purification and titration procedures, using monkey-derived and human-derived cell lines respectively. The REO3 purification methodology has been studied because REO3 is a model virus required in Viral Clearance Studies for clinical trials Phase III. For each purification step different conditions were applied and tested, and all the samples obtained were analyzed. Samples titer and purity were evaluated by titration assay and determination of the residual protein content.