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

Abstract

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.

An Integrated Approach to Aggregate Control for Therapeutic Bispecific Antibodies Using an Improved Three Column Mab Platform‐Like Purification Process

“…Flocculation and precipitation has been used extensively in industry as a means of removing solids and impurities (HCP, DNA and viruses) from a feed stream prior to downstream processing . There are limited reports of aggregate clearance by precipitation.…”

Leveraging single‐pass tangential flow filtration to enable decoupling of upstream and downstream monoclonal antibody processing

“…The bioreactors were operated in fed‐batch mode, using proprietary operating conditions and media for durations of 14–17 days. At the end of production, the cell culture temperature was reduced to 2–12°C, prior to pretreatment using 25% acetic acid to adjust the bioreactor to a target of pH 4.8, to improve clarification . The pretreated cell culture was processed through a Westfalia CSC‐6 separator (Northvale, NJ) to remove the cells and flocculated impurities/debris.…”

Evaluation of predictive tools for cell culture clarification performance

“…To improve the clarification performance, cell culture is often pretreated with flocculating or precipitating agents (Kempken et al, ; McNerney et al, ; Pampel et al, ; Roush and Lu, ). Several studies have shown that flocculation or precipitation can improve the efficiency of the centrifugation step and increase clarification throughput on the subsequent depth filter (Hutchinson et al, ; Kim et al, ; Lydersen et al, ; Lutz et al, ; Roush and Lu, ; Wisniewski and Grasmick, ; Westoby et al, ; Yavorsky et al, ). Cell culture precipitation approaches include treatment with low pH, caprylate, and calcium phosphate.…”