Perfusion processes in mammalian cell cultivations are currently experiencing a renaissance, and are making their way into modern single-use bioreactor systems. In this article, the authors review the common challenges and main applications for perfusion processes. The authors present experimental evidence for reaching extreme cell densities of 150 ×106 cells/mL in an easy to use, high-performance, single- use bioreactor that provides an efficient way to generate cell mass for cell banking or seed trains, as well as production of considerable amounts of monoclonal antibodies (mAbs), even from cell lines with mediocre expression levels.
Recently, there has been renewed interest in perfusion processes. In perfusion culture, fresh medium is continuously fed to the culture while the cells are retained in the bioreactor, and metabolic waste products are removed. Depending on the chosen cell retention device, the large molecular product can either be retained in the bioreactor (concentrated fed-batch) or removed from the culture (perfusion). The latter production mode is typically used when the recombinant protein is prone to degradation, toxic, or inhibitory to the cells. In perfusion processes, cell densities are typically 5– to 10–fold higher than in batch or fed-batch processes. Process optimization of perfusion processes is intended to maintain cell-specific productivity by optimizing the composition of the perfused medium and/or applying a certain cell bleed stream to control cell density and growth rate. It is then possible to achieve 5 to 10 times higher total amount of product compared with batch and fed-batch (3). An economic comparison of the different process modes has recently been published (4). Due to significantly increased productivity, a number of companies have identified concentrated fed-batch and perfusion as tools to reduce production scale and ultimately make commercial production amenable to single use bioreactors. This growing interest in continuous perfusion is further fueled by the increased number of recombinant therapeutic proteins in the development pipeline.
Several cell-retention devices have been developed during the past 20 years. Today, there are a handful of systems that have proven suitable for suspension cell culture at process scale: continuous centrifugation, inclined settlers, hydrocyclones, and alternating tangential filtration (3, 4).
In addition to improved yields in protein production, high cell-density culture also offers advantages for seed production by reducing the bioreactor scale and number of steps necessary to generate the seed for the final production bioreactor. A 10–fold increase in cell density reduces the required inoculum volume by the same factor. To increase flexibility and reduce the time from thawing a cell bank vial to inoculation of the production bioreactor, studies have been performed examining the use of large volume, cryopreserved cultures in cryobags, where the concentrated seed culture has been produced using a perfusion process in order to replace shake flask technology (5, 6). This approach not only reduces the number of bioreactors and steps involved, but also the necessary footprint for seed bioreactors, and investment in equipment.
CHALLENGES WITH CONVENTIONAL CELL-RETENTION SYSTEMS
Perfusion often requires expensive investment in cell-retention equipment, certain infrastructure, and personnel with specialised skills. Therefore, it has not been widely adopted for the production of proteins that are used for research purposes, such as target proteins or therapeutic lead proteins used in early preclinical research. At the same time, early protein supply is often characterised by low expression rates of the cell lines used and the need to produce many different proteins in a given period of time to fuel the development pipeline. We have therefore developed a novel, easy to use, benchtop perfusion bioreactor that is based on rocking motion agitation (BIOSTAT CultiBag RM 20 perfusion, Sartorius Stedim Biotech). In this study, we present data on the high cell-density culture of a mAb-producing Per.C6 model cell line.