Process intensification to produce a difficult-to-express therapeutic enzyme by high cell density perfusion or enhanced fed-batch.

Abstract

Intensified bioprocesses have caught industrial interest in the field of biomanufacturing in recent years. Thanks to new technology, intensified processes can support high cell densities, higher productivities and longer process times, which together can offer lower cost of goods. In this study two different intensified process modes, high cell density perfusion and enhanced fed-batch, were evaluated and compared with a conventional fed-batch process for a difficult-to-express therapeutic enzyme. The intensified process modes were cultivated with a target cell density of 100 × 106 cells/ml and with alternating tangential flow filtration, ATF, as cell retention device. The processes were designed to resemble an established optimized fed-batch process using the knowledge of this process without new dedicated optimization for the intensified modes. The design strategy included decision of the ratio of feed concentrate to base medium and glucose supplementation, which were based on target cell-specific consumption rates of key amino acids and glucose, using a targeted feeding approach (TAFE). A difficult-to-express therapeutic enzyme with multiple glycosylation sites was expressed and analyzed in the different production processes. The two new intensified processes both achieved 10 times higher volumetric productivity (mg/L/day) with retained protein quality and minor changes to the glycan profile compared to the fed-batch process. The study demonstrates the potential of using intensified processes for sensitive complex enzymes. It is shown here that it is possible to transfer a developed fed-batch process into high cell density processes either in intensified fed-batch or steady-state perfusion without new dedicated optimization. The results demonstrated as well that these intensified modes significantly increase the productivity while maintaining the desired product quality, for instance the same amount of product was obtained in 1 day during the perfusion process than in a whole fed-batch run. Without any prior optimization of the perfusion rate, the high cell density perfusion process resulted in only 1.2 times higher medium cost per gram produced protein.