Studies on N-glycosylation of therapeutic Fc-fusion protein in recombinant CHO cells under stressful culture conditions

Abstract

Chinese hamster ovary (CHO) cells are the most widely used mammalian host for producing therapeutic glycoprotein because of their ability to produce proteins with post-translational modifications suitable for clinical use. Glycosylation can affect the efficiency, bioactivity, solubility, and in-vivo stability of a glycoprotein. One of the main challenges in bioprocess development for therapeutic glycoprotein is to maintain high productivity while also obtaining good quality. Therefore, to achieve a consistent glycosylation profile (reduction of glycosylation heterogeneity) in the production of therapeutic proteins, a detailed understanding of the glycosylation biosynthetic pathway in the bioprocessing is necessary. To understand the effects of hyperosmolality on protein glycosylation, recombinant CHO cells producing the Fc-fusion protein were cultivated in the hyperosmolar medium. The hyperosmotic culture increased specific Fc-fusion protein productivity but decreased the proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. Expression of 52 N-glycosylation-related genes was assessed by the NanoString nCounter system. N-linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary N-linked glycans decreased in hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture increased the proportion of highly sialylated and tetra-antennary N-linked glycans, while it increased the expression of the N-glycan branching/antennary genes. Taken together, the results obtained in this study provide a better understanding of the effects of hyperosmolality on N-glycosylation biosynthesis pathway in rCHO cells. Acidic Golgi pH plays an important role in protein glycosylation, one of the critical quality attributes of therapeutic proteins. To determine the intracellular Golgi pH during culture, stable CHO cell clones expressing pHluorin2, a ratiometric pH-sensitive fluorescent protein (FP), in the cis- and trans-Golgi, were constructed by fusing pHluorin2 with specific targeting proteins. Stable CHO cell clones expressing pHluorin2 in the cytoplasm were also constructed. The subcellular localization of FPs was confirmed by immunofluorescence analysis. Live-cell imaging revealed that the intracellular pH of clones expressing the ratiometric pH-sensitive FPs converged to a specific pH range (cis-Golgi: 6.4 – 6.5, trans-Golgi: 5.9 – 6.0, and cytoplasm: 7.1 – 7.2). The intracellular pH was successfully evaluated in various culture conditions. Although culture pH was maintained at 7.2 in a bioreactor, the Golgi pH increased with culture time. Elevated ammonia concentration and osmolality were partially responsible for the increased Golgi pH during bioreactor cultures. Taken together, the application of ratiometric pH-sensitive FPs in monitoring the Golgi pH of CHO cells during culture provides a new perspective to improve protein glycosylation through intracellular pH control.