Use of scanning electron microscopy and energy dispersive X-ray spectroscopy to identify key fouling species during alternating tangential filtration.

Alternating tangential flow filtration (ATF) has become one of the primary methods for cell retention and clarification in perfusion bioreactors. However, membrane fouling can cause product sieving losses that limit the performance of these systems. This study used scanning electron microscopy and energy dispersive X-ray spectroscopy to identify the nature and location of foulants on 0.2 µm polyethersulfone hollow fiber membranes after use in industrial Chinese hamster ovary cell perfusion bioreactors for monoclonal antibody production. Membrane fouling was dominated by proteinaceous material, primarily host cell proteins along with some monoclonal antibody. Fouling occurred primarily on the lumen surface with much less protein trapped within the depth of the fiber. Protein deposition was also most pronounced near the inlet/exit of the hollow fibers, which are the regions with the greatest flux (and transmembrane pressure) during the cyclical operation of the ATF. These results provide important insights into the underlying phenomena governing the fouling behavior of ATF systems for continuous bioprocessing.

Performance of high intensity fed-batch mammalian cell cultures in disposable bioreactor systems.

The adoption of disposable bioreactor technology as an alternate to traditional nondisposable technology is gaining momentum in the biotechnology industry. Evaluation of current disposable bioreactors systems to sustain high intensity fed-batch mammalian cell culture processes needs to be explored. In this study, an assessment was performed comparing single-use bioreactors (SUBs) systems of 50-, 250-, and 1,000-L operating scales with traditional stainless steel (SS) and glass vessels using four distinct mammalian cell culture processes. This comparison focuses on expansion and production stage performance. The SUB performance was evaluated based on three main areas: operability, process scalability, and process performance. The process performance and operability aspects were assessed over time and product quality performance was compared at the day of harvest. Expansion stage results showed disposable bioreactors mirror traditional bioreactors in terms of cellular growth and metabolism. Set-up and disposal times were dramatically reduced using the SUB systems when compared with traditional systems. Production stage runs for both Chinese hamster ovary and NS0 cell lines in the SUB system were able to model SS bioreactors runs at 100-, 200-, 2,000-, and 15,000-L scales. A single 1,000-L SUB run applying a high intensity fed-batch process was able to generate 7.5 kg of antibody with comparable product quality.

Effects of solution environment on mammalian cell fermentation broth properties: enhanced impurity removal and clarification performance.

The processing of recombinant proteins from high cell density, high product titer cell cultures containing mammalian cells is commonly performed using tangential flow microfiltration (MF). However, the increased cellular debris present in these complex feed streams can prematurely foul the membrane, adversely impacting MF capacity and throughput. In addition, high cell density cell culture streams introduce elevated levels of process-related impurities, which increase the burden on subsequent purification operations to remove these complex media components and impurities. To address this challenge, an evaluation of mammalian cell culture broth buffer properties was examined to determine if enhanced impurity removal and clarification performance could be achieved. A framework is presented here for establishing optimized mammalian cell culture buffer conditions, involving trade-offs between product recovery and purification and improved clarification at manufacturing-scale production. A reduction in cell culture broth pH to 4.7-5.0 induced flocculation and impurity precipitation which increased the average feed particle-size. These conditions led to enhanced impurity removal and improved MF throughput and filter capacity for several mammalian systems. Feed conditions were further optimized by controlling ionic composition along with pH to improve product recovery from high cell density/high product titer cell cultures.