Author(s): Cunha, Bárbara Martins Paiva da
Date: 2016
Persistent ID: http://hdl.handle.net/10362/20208
Origin: Repositório Institucional da UNL
Subject(s): Cell therapy; Downstream processing; Filtration; Stem cells; Process development; Cell characterization; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias
During the past decades, human stem cells (hSC) have emerged as a therapeutic alternative for several diseases. However, their clinical transfer still faces major challenges. Amongst them are the production of high cell amounts, the delivery of those high cell numbers at low volumes with minimal presence of residuals, while assuring the desired phenotype and function/potency. Although substantial efforts have been placed on the large-scale production of hSC and/or derivatives, the development of scalable approaches for downstream processing (DSP) of hSC and the establishment of fully integrated platforms for cell manufacturing has been long awaited. The goal of this PhD project was to establish scalable and integrated DSP strategies for the clarification, volume reduction (concentration) and washing unit operations for humanderived adult and pluripotent SC. Human mesenchymal stem cells (hMSC) and human induced pluripotent stem cells (hiPSC) were used as challenging and complex cell-based products. Combining membrane technology and chromatographic tools, a flexible platform based on cost-effective, robust, scalable, and compatible with current good manufacturing practices (cGMP) processes, compliant with different stem cell types and that can be transferred to clinical/industrial settings was developed. Initially, it was evaluated the applicability of filtration methodologies, as dead end filtration and tangential flow filtration, for the clarification and concentration of hMSC, respectively. Different process parameters and their impact on hMSC quality were studied. Polypropylene filters with pore sizes higher than 75 μm ensured the efficient removal of microcarriers from the cell suspension bulk, without compromising cells’ recovery yields or viability. Furthermore, hMSC could be successfully concentrated up to a factor of 10 while maintaining their identity, differentiation capacity and high cell viability, allowing for the recovery of over 80% of viable cells; an initial cell concentration higher than 2 x 105 cell/mL, and polysulfone membranes with pore sizes higher than 0.45 μm were identified to be key conditions to obtain such concentration factors; shear rate and permeate flux were also shown to impact cells’ recovery yields, viability and quality. The applicability of the previously developed TFF methodologies to hiPSC processing, was assessed applying a Design of Experiments (DoE) approach. The impact of the process parameters of the volume reduction unit operation on hiPSC’s recovery, viability, expression of pluripotency markers and pluripotency differentiation potential was assessed and compared with results previously attained with hMSC. A mathematical model of the process was designed and after understanding how the shear rate, permeate flux and cell load factors interacted between them, the condition that maximized cell recovery and viability showed to be similar between both stem cell types. A robustness analysis was performed and the success rate of these operating conditions was assessed (65 - 70%). A parametric study was then conducted, identifying that increasing the shear rate (up to 3370 s-1) allowed to achieve the specified requirements for cell recovery yield (> 80%) and viability (> 90%) in 100% of the cases and no impact in hiPSC’s proliferation capacity, expression of pluripotency markers and differentiation potential was observed.(...)
