Molecular biology technology for the generation of therapeutic monoclonal antibodies (mAbs) has rapidly developed, and the number of antibodies approved is increasing every year (Tsumoto et al., 2019, Zhang et al., 2022). Therapeutic mAbs are generally produced by the Chinese hamster ovary (CHO) cells and have been approved for the treatment of cancer and autoimmune diseases (Tsumoto et al., 2019, Zhang et al., 2022). Consistent manufacturing that regulates critical quality attributes (CQAs) is required to ensure the safety and efficacy of therapeutic mAbs (Madabhushi et al., 2021). Notably, N-linked glycosylation found at the Asn297 of Fc region has been recognized as a CQA because of its therapeutic efficacy (antibody-dependent cellular cytotoxicity: ADCC and complement-dependent cytotoxicity: CDC), pharmacokinetics/pharmacodynamics, and immunogenicity of mAbs, which depend on the structure of N-linked glycans (Madabhushi et al., 2021, Wang et al., 2020). N-linked glycans consist of a constant core region of two N-acetylglucosamine (GlcNAc) and three mannose (Man) units, with additional saccharides, including fucose (Fuc), GlcNAc, galactose (Gal), and sialic acid (N-acetylneuraminic acid: Neu5Ac and Neu5Gc). The heterogeneity of the N-linked glycan profiles of mAbs can be attributed to the different numbers and linkages of additional saccharides. Furthermore, the structure of N-linked glycans affects the characteristics of mAbs. For instance, ADCC is influenced by the fucosylation (Shields et al., 2002, Shinkawa et al., 2003) and galactosylation (Kiyoshi et al., 2018, Thomann et al., 2016) of N-linked glycans. CDC is also affected by the galactosylation (Peschke et al., 2017) and sialylation (Quast et al., 2015) of N-linked glycans. A high-mannose structure may affect the rapid clearance or ADCC activity (Kanda et al., 2007, Zhou et al., 2008). Non-human epitopes, such as Neu5Gc and Galα1–3 Gal at the terminal of N-linked glycan found in NS0, a murine myeloma cells, exhibit immunogenicity (Ghaderi et al., 2012, Kim et al., 2020, Villacrés et al., 2021). Despite the fact that CHO cells can also manufacture Galα1–3 Gal epitopes in proteins (Bosques et al., 2010), CHO cells have been widely employed for mAb production because the glycotype of mAbs from CHO cells is similar to that of human serum antibodies (Zhang et al., 2022). However, the heterogeneity of the N-linked glycan profile of mAbs depends on the cell culture duration and changes in sugar nucleotides and glycosylation enzyme levels (Gramer et al., 2011, Madabhushi et al., 2021). Glucose limitation and culture duration result in reduced mAb production and increased galactosylation (Fan et al., 2015). Supplementation of the combination of uridine, manganese (Mn2+), and galactose modulate antibody galactosylation (Gramer et al., 2011, Villacrés et al., 2021). Increased specific productivity (qp) of mAbs by cell culturing in media under high osmolarity or supplementation with sodium butyrate results in the reduction of galactosylation and an increase in mannosylation, while decreased qp by cell culturing in media under high pH conditions results in increased galactosylation (Madabhushi et al., 2021). These findings related to cell culture have contributed to the manufacturing and quality control of mAbs. However, different manufacturing periods and/or manufacturers affect the N-linked glycan profile and resultant ADCC activity of biosimilars (Xie et al., 2020, Yao et al., 2022). Thus, further fundamental studies on the regulation of mAb glycosylation profiles are required.

Polyamines (putrescine, PUT; spermidine, SPD; and spermine, SPM) are present in millimolar concentrations in all living organisms and play essential roles in normal cell growth and differentiation (Igarashi and Kashiwagi, 2018, Nakanishi and Cleveland, 2021). PUT, SPD, and SPM contained two, three, and four amino groups, respectively (Supplementary Fig. S1). PUT is synthesized from ornithine (ORN) by ornithine decarboxylase (ODC), a rate-limiting enzyme in the polyamine biosynthesis pathway. SPD is synthesized from putrescine by spermidine synthase, and spermine is synthesized from spermidine by spermine synthase. Intracellular polyamine levels are regulated at various steps, including synthesis, degradation, and transport (Casero et al., 2018, Igarashi and Kashiwagi, 2010), and are affected by external stimuli, aging, and diseases. For example, the activation of ODC activity and the resulting increase in intracellular polyamine levels were observed during the differentiation of rabbit costal chondrocytes induced by parathyroid hormone (Takano et al., 1987, Takano et al., 1983) and bovine lymphocytes exposed to concanavalin A (Kakinuma et al., 1988). In contrast, tissue polyamine levels decrease during the aging process in animals (Al-Habsi et al., 2022, JAENNE et al., 1964, Nishimura et al., 2006). Polyamines exist mostly as polyamine-RNA complexes (Watanabe et al., 1991), and cell growth stimulation by polyamines is due to protein synthesis enhancement of specific genes related to cell growth (Igarashi and Kashiwagi, 2021). In Escherichia coli, the expression of 20 proteins, including transcription and translation factors, is stimulated by polyamines at the translational level (Igarashi and Kashiwagi, 2018). Thus far, these genes are regulated by inefficient translation systems, and polyamines interact with specific mRNA, tRNA, and ribosomal RNA to facilitate their translation. In eukaryotic cells, genes whose expression levels are enhanced by polyamines at the translational level have been explored to understand the physiological function of polyamines, and 13 genes have been identified to date (Igarashi and Kashiwagi, 2021). We found that the expression of exostosin glycosyltransferase 2 (EXT2) and chondroitin sulfate synthase 1 (CHSY1) was enhanced by polyamines at the translational level, thereby stimulating the maturation of heparan sulfate (HS) and chondroitin sulfate (CS), which are members of the glycosaminoglycan family (Imamura et al., 2016, Yamaguchi et al., 2018). Although it has been reported that rat intestinal glycoprotein fucosylation is regulated by polyamines (Biol-N′Garagba et al., 2002), the effect of polyamines on N-glycan synthesis is not fully understood.

In this study, the effect of polyamines on the N-linked glycan profile of mAbs in CHO cells was examined. We found that ER stress caused by polyamine depletion stimulates the induction of B4GALT1 mRNA, resulting in the increment of galactosylation of mAbs.