A Universal High Density Cell Respirator (HDCR) Bioreactor for Intensified Production of Gene Therapy Vectors

Abstract

AAV-based gene therapy vectors are under intense investigation and rapidly becoming established as clinical therapy for gene-based diseases including inherited diseases. Despite their great promise, the single biggest limitation to their widespread use is the inability to manufacture sufficient quantities of high quality AAV vectors rapidly particularly at low cost. This represents the single biggest limitation to the development of clinical AAV gene therapy. The intensification of cell-based production processes holds great promise for increasing the capacity and speed of AAV vector manufacturing while also reducing cost, by saving on space, time, labor, and resources. While much effort to date has focused on improving cell specific productivity (vector genomes (vg)/cell), little progress has been made on increasing cell density, which remain at 10⁶⁻⁷ cells/mL due to limitations of either surface area or gas exchange and shear forces (e.g. stirred tank reactors, perfusion reactors). With total productivity being the product of specific cell productivity and cell density, there exists substantial untapped potential in high-density culture. Recognizing the enormous discrepancy in the necessary gaseous exchange rate compared to soluble nutrient/waste exchange rate for most cells (e.g. 90× for HEK293), we hypothesized that 1) decoupling mass transport through novel bioreactor architecture would enable higher cell densities and that 2) the optimized cell niche afforded by this architecture would maintain cell specific productivity even at high cell densities, with an overall impact of significantly elevated production capacity. With the goal of improving AAV vector production capacities and to address issues of cell growth and densities, we designed a high-density cell respirator (HDCR). The HDCR is a scalable bioreactor comprised of stackable, gas and media perfusable membranes, which achieves an oxygen mass transfer coefficient k_La of > 40/hr via membrane permeation to support high density culture >10⁸ cells/mL. The micro-architecture of the membranes has been engineered using integrated finite element modeling to guarantee that all cells receive sufficient gas, nutrient, and waste exchange and are protected from shear forces. We present cell growth curves demonstrating the system is compatible with suspension (e.g. CHO-S), adherent (e.g. HEK293, A549), and microcarrier (e.g. CV-1 on Cytodex-3) cultures to >10⁸ cells/mL making it a universal platform. Importantly, we demonstrate that the HDCR bioreactor is compatible with AAV vector production, including the steps of cell seeding, expansion, transfection, feeding, and harvesting. We show for the first time that the optimized cell growth niche in the HDCR maintains productivity (within 80% vg/cell of low-density culture) despite increasing cell density by 400% for a total increase in AAV production of 320%. The results of ongoing optimization studies for AAV production in the HDCR will be presented. From these initial empirical results and modelling, we predict that a scaled HDCR bioreactor fitting inside a standard 160L tissue culture incubator would support the expansion of >2×10¹² cells and production of up to 10¹⁷ vector genomes of AAV. By matching mass transport with cellular demands AAV production can be intensified, unlocking the potential for researchers to screen AAV candidates faster and for clinical vectors to be affordably manufactured.

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