Differential response in downstream processing of CHO cells grown under mild hypothermic conditions.

The manufacture of complex therapeutic proteins using mammalian cells is well established, with several strategies developed to improve productivity. The application of sustained mild hypothermic conditions during culture has been associated with increases in product titer and improved product quality. However, despite associated cell physiological effects, very few studies have investigated the impact on downstream processing (DSP). Characterization of cells grown under mild hypothermic conditions demonstrated that the stationary phase was prolonged by delaying the onset of apoptosis. This enabled cells to maintain viability for extended periods and increase volumetric productivity from 0.74 to 1.02 g L(-1) . However, host cell proteins, measured by ELISA, increased by ∼50%, attributed to the extended time course and higher peak and harvest cell densities. The individual components making up this impurity, as determined by SELDI-TOF MS and 2D-PAGE, were shown to be largely comparable. Under mild hypothermic conditions, cells were less shear sensitive than those maintained at 37°C, enhancing the preliminary primary recovery step. Adaptive changes in membrane fluidity were further investigated by adopting a pronounced temperature shift immediately prior to primary recovery and the improvement observed suggests that such a strategy may be implementable when shear sensitivity is of concern. Early and late apoptotic cells were particularly susceptible to shear, at either temperature, even under the lowest shear rate investigated. These findings demonstrate the importance of considering the impact of cell culture strategies and cell physiology on DSP, by implementing a range of experimental methods for process characterization.

Impact of aeration strategy on CHO cell performance during antibody production.

Stirred tank bioreactors using suspension adapted mammalian cells are typically used for the production of complex therapeutic proteins. The hydrodynamic conditions experienced by cells within this environment have been shown to directly impact growth, productivity, and product quality and therefore an improved understanding of the cellular response is critical. Here we investigate the sub-lethal effects of different aeration strategies on Chinese hamster ovary cells during monoclonal antibody production. Two gas delivery systems were employed to study the presence and absence of the air-liquid interface: bubbled direct gas sparging and a non-bubbled diffusive silicone membrane system. Additionally, the effect of higher gas flow rate in the sparged bioreactor was examined. Both aeration systems were run using chemically defined media with and without the shear protectant Pluronic F-68 (PF-68). Cells were unable to grow with direct gas sparging without PF-68; however, when a silicone membrane aeration system was implemented growth was comparable to the sparged bioreactor with PF-68, indicating the necessity of shear protectants in the presence of bubbles. The cultures exposed to increased hydrodynamic stress were shown by flow cytometry to have decreased F-actin intensity within the cytoskeleton and enter apoptosis earlier. This indicates that these conditions elicit a sub-lethal physiological change in cells that would not be detected by the at-line assays which are normally implemented during cell culture. These physiological changes only result in a difference in continuous centrifugation performance under high flow rate conditions. Product quality was more strongly affected by culture age than the hydrodynamic conditions tested.

Host cell protein adsorption characteristics during protein A chromatography.

Protein A chromatography is a critical and 'gold-standard' step in the purification of monoclonal antibody (mAb) products. Its ability to remove >98% of impurities in a single step alleviates the burden on subsequent process steps and facilitates the implementation of platform processes, with a minimal number of chromatographic steps. Here, we have evaluated four commercially available protein A chromatography matrices in terms of their ability to remove host cell proteins (HCPs), a complex group of process related impurities that must be removed to minimal levels. SELDI-TOF MS was used as a screening tool to generate an impurity profile fingerprint for each resin and indicated a number of residual impurities present following protein A chromatography, agreeing with HCP ELISA. Although many of these were observed for all matrices there was a significantly elevated level of impurity binding associated with the resin based on controlled pore glass under standard conditions. Use of null cell line supernatant with and without spiked purified mAb demonstrated the interaction of HCPs to be not only with the resin back-bone but also with the bound mAb. A null cell line column overload and sample enrichment method before 2D-PAGE was then used to determine individual components associated with resin back-bone adsorption. The methods shown allow for a critical analysis of HCP removal during protein A chromatography. Taken together they provide the necessary process understanding to allow process engineers to identify rational approaches for the removal of prominent HCPs.