Multivariate analysis of the effects of high seeding density on therapeutic protein production and sialylation in an intensified CHO cell culture process.

Intensified processes in Chinese hamster ovary (CHO) cell culture are increasingly implemented to enhance therapeutic protein production in biopharmaceutical manufacturing; however, their impact on glycan-related product quality attributes, particularly terminal sialylation, remains insufficiently characterized. In this study, a design of experiments approach combined with an Ambr® 250 high-throughput bioreactor system was used to evaluate the effects of key process parameters on cell culture performance and glycan quality in recombinant CHO cells producing an Fc-fusion glycoprotein under intensified conditions. Three factors-seeding density, pH, and dissolved oxygen (DO)-were evaluated using a Box-Behnken design. Their effects on the integral of viable cell density (IVCD), product titer, and tetra-sialylation level were quantified using response surface modeling. Regression analysis identified seeding density and pH as the primary parameters influencing the balance between product titer and glycan maturation, whereas DO showed comparatively minor effects. Increasing seeding density significantly enhanced IVCD and product titer but reduced terminal sialylation, particularly at elevated pH, indicating a trade-off between process intensification and glycan quality. By integrating predefined criteria for product titer and tetra-sialylation, a probabilistic design space was established, and a robust operating condition was identified at pH 6.9 with a moderate seeding density. Experimental validation confirmed balanced protein production and sialylation under this condition, whereas higher seeding densities did not maintain acceptable glycan quality. These findings highlight the importance of multivariate optimization in defining operating ranges that support both high protein production and consistent product quality in intensified CHO cell culture processes.