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Analytical procedures validation for good manufacturing practice (GMP) production of mesenchymal stem cells from different sources

      The production of Mesenchymal stem cells (MSC) for cellular therapy should fulfil the requirements of GMP rules. In this contest, the production process and the analytical methods for quality control should be validated to demonstrate standardization and compliance with quality requisites. In our hospital-based GMP facility we produce MSC from different sources and we developed and validated a set of quality control assays for final products: sterility and endotoxins assay (<0,25 EU/mL), absence of mycoplasma and adventitious viruses, cell dose, purity (as percentage of cells CD90+/CD105+/CD45- > 80%) and viability (as percentage of cells PI- > 80%). Sterility was validated following Eu. Ph. 2.6.27 with both the standard microbial strains and those isolated during environmental monitoring. The endotoxin method (Eu. Ph. 2.6.14) was validated at different dilutions of the final product to exclude inhibition. The method to detect mycoplasma was compliant to the Pharmacopoeia requirements (Ph 2.6.7). We also performed a viral validation to exclude the accidentally introduction of respiratory viruses, Cytomegalovirus and Epstein-Barr Virus during production. For cell counting, an automated method (NucleoCounter, Chemometec) was found to be more accurate (R2 > 0.9) and precise (CV < 10%) than a standard manual method (Burker chamber). Moreover, we assessed the repeatability and reproducibility (CV < 5%) of the immunophenotyping analysis of MSC by flow cytometry to determine the purity of our products as the percentage of cells that co-express CD90 and CD105 and is negative to CD45. Finally, cell viability by Annexin-PI analysis was evaluated on MSC after expansion and cryopreservation. All these methods have been included in the Investigational Medicinal Product Dossier of all the MSC-based products currently provided by our Institution in several phase I/II clinical trials already ongoing.
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