Improvement of protein purity in etanercept production through process optimization in recombinant CHO cell-based...

Maintaining high protein purity is crucial for ensuring the efficacy, safety, and productivity of therapeutic glycoproteins. Given that proteins produced within transient gene expression (TGE) systems are ultimately intended for incorporation into production cell lines developed using stable gene expression (SGE) systems, it is crucial to evaluate protein purity across both systems and explore strategies for improvement. In this study, elevated levels of both high- and low-molecular-weight impurities were observed in etanercept (ETN) produced under the evaluated TGE conditions compared with the SGE system. To address these purity concerns, we investigated the effects of various process modifications, including chemical treatments, temperature downshifts, and adjustments to kit components within the TGE system. Results indicated that rapamycin treatment, a temperature downshift to 30 °C, and the omission of Enhancer material increased the monomer proportion of ETN. Combining a temperature downshift to 30 °C with omission of the Enhancer material reduced overall ETN production but increased the monomer ratio to levels comparable to those in the SGE system. Moreover, the combination of rapamycin treatment, a temperature downshift to 30 °C, and an extended culture duration significantly enhanced both total and monomer ETN production while maintaining higher purity. These improvements were similarly observed in the HEK293 cell-based TGE system, demonstrating that the optimized culture conditions possess broad applicability across mammalian TGE systems. These findings demonstrate that comprehensive optimization of culture parameters can significantly enhance protein purity in TGE-based protein production, enabling purity levels comparable to those obtained from SGE systems under the conditions evaluated in this study.