Perfusion Cell Culture Decreases Process and Product Heterogeneity in a Head-to-Head Comparison With Fed-Batch.

In this study, the authors compared the impacts of fed-batch and perfusion platforms on process and product attributes for IgG1- and IgG4-producing cell lines. A "plug-and-play" approach is applied to both platforms at bench scale, using commercially available basal and feed media, a standard feed strategy for fed-batch and ATF filtration for perfusion. Product concentration in fed-batch is 2.5 times greater than perfusion, while average productivity in perfusion is 7.5 times greater than fed-batch. PCA reveals more variability in the cell environment and metabolism during the fed-batch run. LDH measurements show that exposure of product to cell lysate is 7-10 times greater in fed-batch. Product analysis shows larger abundances of neutral species in perfusion, likely due to decreased bioreactor residence times and extracellular exposure. The IgG1 perfusion product also has higher purity and lower half-antibody. Glycosylation is similar across both culture modes. The first perfusion harvest slice for both product types shows different glycosylation than subsequent harvests, suggesting that product quality lags behind metabolism. In conclusion, process and product data indicate that intra-lot heterogeneity is decreased in perfusion cultures. Additional data and discussion is required to understand the developmental, clinical and commercial implications, and in what situations increased uniformity would be beneficial.

The effects of alternating tangential flow (ATF) residence time, hydrodynamic stress, and filtration flux on high-density perfusion cell culture.

In this study, we investigated the effects of alternating tangential flow (ATF) cell separation on high-density perfusion cultures. We have developed methods to estimate theoretical residence times of cells in the ATF system and discovered that long residence times (above 75 s) correlate with decreased growth, metabolism, and productivity. We have calculated energy dissipation rates in the ATF transfer line and filter and empirically studied the impacts of increased exchange rates on cell culture, determining that increased hydrodynamic stress can lead to decreased cell size, lactate production, and specific productivity. Finally, we have conducted experiments to understand the relationship between filtration fluxes and ATF membrane fouling, finding that at fluxes above 60 L·m-2 ·day -1 , protein sieving coefficients see significant rates of decrease (greater than 1% per day). While most of these studies have been conducted with one cell line at one target viable cell density (40 million cells/ml), the general, directional knowledge arising from this study should be applicable to other conditions and programs, ultimately leading to more robust and well-designed perfusion processes.

Integrated continuous production of recombinant therapeutic proteins.

In the current environment of diverse product pipelines, rapidly fluctuating market demands and growing competition from biosimilars, biotechnology companies are increasingly driven to develop innovative solutions for highly flexible and cost-effective manufacturing. To address these challenging demands, integrated continuous processing, comprised of high-density perfusion cell culture and a directly coupled continuous capture step, can be used as a universal biomanufacturing platform. This study reports the first successful demonstration of the integration of a perfusion bioreactor and a four-column periodic counter-current chromatography (PCC) system for the continuous capture of candidate protein therapeutics. Two examples are presented: (1) a monoclonal antibody (model of a stable protein) and (2) a recombinant human enzyme (model of a highly complex, less stable protein). In both cases, high-density perfusion CHO cell cultures were operated at a quasi-steady state of 50-60 × 10(6) cells/mL for more than 60 days, achieving volumetric productivities much higher than current perfusion or fed-batch processes. The directly integrated and automated PCC system ran uninterrupted for 30 days without indications of time-based performance decline. The product quality observed for the continuous capture process was comparable to that for a batch-column operation. Furthermore, the integration of perfusion cell culture and PCC led to a dramatic decrease in the equipment footprint and elimination of several non-value-added unit operations, such as clarification and intermediate hold steps. These findings demonstrate the potential of integrated continuous bioprocessing as a universal platform for the manufacture of various kinds of therapeutic proteins.