Scalable manufacturing of anchorage-dependent cells for allogeneic cell therpies: current limitations and an innovative solution

Abstract

Current cell culture processes for allogeneic cell therapies widely utilize a 2D approach, where anchorage-dependent cells are grown on flat, plastic plates. While stacking plates can achieve a certain level of scale-up adequate for clinical production, scale-up to commercial manufacturing using this 2D approach is not operationally and economically feasible. Therefore an alternative 3D process, where cells are grown on microcarriers in suspension, is considered to be a viable option. However, this 3D approach has challenges to scale up in conventional stirred bioreactors, presumably due to the shear stress associated with high speed mixing. Novel single-use bioreactors with Vertical-Wheel technology have recently been introduced. These bioreactors promote efficient, homogenous liquid mixing and microcarrier suspension at lower agitation speeds. Computational fluid dynamics analysis indicates that the low shear environment inside these vessels is maintained across the full range of working volumes, from 0.1 to 500 liters. Human bone marrow-derived mesenchymal stem cells (hBM-MSC) were grown in Vertical-Wheel bioreactors without the use of anti-foaming agents or shear protectants. By optimizing culture conditions, including microcarriers, culture media, agitation speeds, and other operating parameters, a final hBM-MSC density exceeding 2x10cells/mL was achieved. Correlations between cell growth, microcarrier concentration, and Kolmogorov scales were also found, which can be applied to predict appropriate process conditions during scale-up. Other process conditions such as dynamic seeding conditions, media exchange, and in-reactor cell harvest for recovery from microcarriers were also evaluated. Relevant cell quality assays were used to address maintenance of cell quality during process scale-up. This study show the potential of Vertical-Wheel bioreactors as a viable manufacturing platform for the commercial production of allogeneic cell therapies.