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. 2026 Mar 13;110:104. doi: 10.1007/s00253-026-13778-1

Effects of sialylated oligosaccharides on the N- and O-glycosylation of etanercept in recombinant CHO cells

Tae-Ho Kim 1,2,#, Hoon-Min Lee 1,3,#, So-Hui Ryu 1,4, Hyun-Seung Kim 1,3, Seung Jin Koo 1,3, Mi-Jung Kang 1, Jungmok You 2,4,✉, Yeon-Gu Kim 1,3,✉
PMCID: PMC12988902  PMID: 41826544

Abstract

Abstract

In mammalian cell-based bioprocesses, glycosylation of recombinant therapeutic proteins requires stringent control and monitoring as it may affect therapeutic efficacy and safety. The sialylation of glycosylated moieties plays an important role in determining the in vivo clearance of therapeutic proteins by extending their serum half-life. In this study, six enzymatically synthesized oligosaccharides (3′-sialyllactose, 6′-sialyllactose, sialyllacto-N-tetraose a, disialyllacto-N-neotetraose, lacto-N-tetraose, and lacto-N-neotetraose) and lactose were evaluated for their effectiveness on the N- and O-glycan profiles of etanercept, a therapeutic Fc-fusion glycoprotein consisting of 3 N- and 13 O-glycosylation sites, produced in recombinant Chinese hamster ovary (rCHO) cells. Of these seven compounds, only sialylated oligosaccharide supplementation increased the proportion of the acidic isoforms, di-sialylated N-glycan and di-sialylated O-glycan, as well as the total sialic acid content of etanercept, although the degree of the effect varied. Increased sialylation resulted from increased concentrations of intracellular CMP-sialic acid, with the highest increase observed for disialyllacto-N-neotetraose, which contains the most di-sialic acid residues. In contrast, four sialylated oligosaccharides did not enhance the sialylation of etanercept produced by human embryonic kidney 293 (HEK293) cells. Taken together, enzymatically synthesized sialylated oligosaccharides represent novel supplements to enhance the sialylation of therapeutic Fc-fusion glycoproteins in rCHO cell culture.

Key points

• Sialylated oligosaccharides supplementation in rCHO cell culture increased the proportion of di-sialylated N- and O-glycans of Fc-fusion glycoprotein.

• Among the sialylated oligosaccharides, disialyllacto-N-neotetraose (DSLNnT) increased intracellular CMP-SA the most in two rCHO cells.

• The effect of sialylated oligosaccharides on sialylation was also evaluated in HEK293 cells; however, no significant enhancement was observed.

Graphical abstract

graphic file with name 253_2026_13778_Figa_HTML.webp

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Supplementary Information

The online version contains supplementary material available at 10.1007/s00253-026-13778-1.

Keywords: Sialylated oligosaccharides, Recombinant Chinese hamster ovary cells, N-Glycosylation, O-Glycosylation, Etanercept

Introduction

Controlling and optimizing the glycosylation profile of recombinant therapeutic proteins is a major goal in the biopharmaceutical manufacturing process. Glycosylation patterns may be affected by various factors, including the expression of glycosylation-related genes, perturbations in culture conditions, such as pH, dissolved oxygen, and temperature, and the characteristics of the host cells (Ha et al. 2022; Naik et al. 2018; Wong et al. 2010b). These variations result in the heterogeneity of glycosylation profiles, which have a significant effect on the biological function of the resulting therapeutic proteins (Hossler et al. 2009). Therefore, obtaining a consistent glycosylation profile is essential to meet regulatory agency demands, as glycosylation is considered an important quality attribute of recombinant therapeutic proteins produced in mammalian cells.

Recombinant Chinese hamster ovary (rCHO) cells are the most widely used mammalian cells for the production of recombinant therapeutic proteins because of their ability to produce human-like posttranslational modifications (Lalonde and Durocher 2017). A major challenge in the production process using rCHO cells is the balance of achieving the desired glycosylation profile with high productivity. For recombinant sialylated glycoproteins, sialylation can be impaired by intracellular or extracellular factors. In fed-batch cultures, factors such as oxygen transfer limitations, ammonia accumulation, and increased osmotic pressure can alter the expression of genes involved in nucleotide sugar synthesis and N-glycosylation, thereby reducing the sialic acid content of recombinant proteins (Ha et al. 2015; Lee et al. 2017a; Lewis et al. 2016; Wong et al. 2010a). In addition to intracellular events, extracellular sialidase released from dead cells may affect the final sialylation level (Gramer et al. 1995; Zhang et al. 2010). Reduced sialylation has a greater impact on the half-life of highly sialylated glycoproteins, including cytokines and Fc-fusion glycoproteins, compared with that in monoclonal antibody (Liu 2015). Thus, several strategies have been undertaken to improve the sialylation of glycoproteins produced in rCHO cell cultures, such as the addition of nucleotide sugar precursors and glycoengineering of rCHO cell lines (Blondeel and Aucoin 2018; Tejwani et al. 2018).

Supplementing cultures with nucleotide sugar precursors is a simple method for regulating glycosylation. By increasing the concentration of intracellular nucleotide sugars, the final glycan profile of the recombinant protein can be influenced (Naik et al. 2018). The glycan structure of glycoproteins produced in mammalian cells is predominantly a complex type of N-glycan, which is matured to the final structure of the glycan by the sequential attachment of N-acetylglucosamine, galactose, and sialic acid to the core structure (Fisher et al. 2019). To facilitate the attachment of these monosaccharides to glycans by increasing the availability of intracellular nucleotide sugars, precursors, such as glucosamine, galactose, and N-acetylmannosamine (ManNAc), are widely used as supplements (Loebrich et al. 2019; Wong et al. 2010b). In particular, the addition of galactose and ManNAc can enhance the sialylation of glycoproteins produced in rCHO cells (Gu and Wang 1998; Wong et al. 2010b). The addition of galactose, either alone or in combination with nucleosides, such as uridine, was shown to increase the sialylation of glycoproteins, including TNFR-Fc and interferon-gamma (Liu et al. 2015; Wong et al. 2010b). Furthermore, previous studies have shown that ManNAc supplementation enhances the sialylation of various recombinant glycoproteins produced by rCHO cells and increases the intracellular CMP-sialic acid (CMP-SA) concentration (Cha et al. 2018; Lalonde et al. 2020; Lee et al. 2017b).

In addition to the conventional methodology of using ManNAc to enhance sialylation in rCHO cell culture, the development of various chemical replacements with similar effects is needed, considering the clonal variation encountered with rCHO cells. Sialyllactose, which consists of lactose linked to sialic acid, is the most abundant sialylated oligosaccharide present in human milk (Yang et al. 2019). Our previous study demonstrated that supplementation with enzymatically synthesized 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL) enhanced the sialylation of Fc-fusion glycoproteins in rCHO cell culture (Lee et al. 2024). This enhanced sialylation was attributed to an increase in intracellular CMP-SA concentration, and the extent of increased sialylation was comparable to or slightly better than that of ManNAc (Lee et al. 2024). Based on these findings, examining the potential for novel applications of various sialylated oligosaccharides in rCHO-based production processes is warranted to increase the sialylation of glycoproteins.

In this study, we established the N- and O-glycan profiles of etanercept, a therapeutic Fc-fusion glycoprotein containing 3 N- and 13 O-glycans produced by two rCHO cell lines following supplementation with enzymatically synthesized sialylated oligosaccharides. We assessed the isoform distribution, N- and O-glycan profiles, and total sialic acid content of etanercept using established analytical methods, including isoelectric focusing (IEF) gel analysis, hydrophilic interaction chromatography (HILIC), and ultra-high-performance liquid chromatography (UHPLC)-based sialic acid quantitation, as described previously (Lee et al. 2024). The concentration of the nucleotide sugars was also quantified by ion-pair reversed-phase HPLC. Furthermore, the effect of sialylated oligosaccharides on glycosylation was assessed in human embryonic kidney 293 (HEK293) cells producing etanercept.

Materials and methods

Cell lines and cell culture maintenance

The coding sequences of etanercept and dihydrofolate reductase (DHFR) were obtained from a plasmid used in previous studies (Park et al. 2023, 2021). The PCR-amplified etanercept and DHFR fragments were subcloned into the pcDNA™ 3.1/Zeo(+) vector (Thermo Fisher Scientific, Waltham, MA, USA), yielding the pcDNA3.1/Zeo/DHFR/etanercept expression vector. Stable rCHO cells producing etanercept were established by transfecting this vector encoding etanercept and DHFR into ExpiCHO-S™ cells (Thermo Fisher Scientific) and selected with stepwise increases in the concentration of methotrexate (MTX) (Sigma-Aldrich, St. Louis, MO, USA) from 1 to 20 nM and 500 μg/mL G418 (Thermo Fisher Scientific). Two stable clones were selected with relatively low (CHO-ETN-L clone) and high (CHO-ETN-H clone) levels of sialylation of etanercept. These two clones were cultivated in serum-free suspension culture using PowerCHO®−2 medium (Lonza, Verbier, Belgium) supplemented with 4 mM glutamine and 20 nM MTX.

Stable HEK293 cells producing etanercept were established by transfecting a vector encoding etanercept into Expi293F™ cells (Thermo Fisher Scientific) and selected for neomycin resistance with 400 μg/mL G418 (Thermo Fisher Scientific). The stable clone, HEK293-ETN clone, was selected as a higher yield of etanercept and maintained in a serum-free suspension culture using EX-CELL® 293 Serum-Free Medium (SAFC Biosciences, Hampshire, UK) containing 4 mM glutamine. The two rCHO cell lines and one HEK293 cell line producing etanercept were maintained in a Climo-shaking CO2 incubator (Adolf Kühner, Birsfelden, Switzerland) at 37℃, 110 rpm, 70% humidity, and 5% CO2.

Cell culture and oligosaccharide supplementation

For etanercept production, the CHO-ETN-L and CHO-ETN-H clones in an exponential growth phase were inoculated at a concentration of 3.0 × 105 cells/mL into 125 mL Erlenmeyer flasks (Corning, Corning, NY, USA) containing 30 mL of PowerCHO®−2 medium with 4 mM glutamine and 20 nM MTX. The HEK293-ETN clone in the exponential growth phase was also inoculated at a concentration of 5.0 × 105 cells/mL into 125 mL Erlenmeyer flasks containing 30 mL of EX-CELL® 293 medium with 4 mM glutamine.

Six enzymatically-synthesized oligosaccharides, 3′-SL, 6′-SL, sialyllacto-N-tetraose a (LSTa), disialyllacto-N-neotetraose (DSLNnT), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT), were obtained as a powder from GeneChem Inc. (Daejeon, Republic of Korea). Lactose was purchased from Sigma-Aldrich. Six enzymatically synthesized oligosaccharides and lactose were added individually to each culture at a concentration of 10 mM on day 3, and the culture without oligosaccharides was used as a control. Culture samples were collected daily to determine the cell concentration, viability, and etanercept concentration. After centrifugation, the culture supernatants were aliquoted and stored at − 70℃ until further analysis.

Cell concentration, viability, and etanercept concentration assay

Cell concentration and viability were measured using a Cedex HiRes analyzer (Roche Diagnostics, Basel, Switzerland) with the trypan blue dye exclusion method. The etanercept concentration in the culture supernatant was quantitated using a Cedex Bio analyzer (Roche Diagnostics) following the manufacturer’s instructions.

Purification of etanercept and IEF gel analysis

Etanercept was purified from the cell culture supernatants by protein A chromatography using HiScreen™ Fibro PrismA (Cytiva, Little Chalfont, UK) and a liquid chromatography system (ÄKTA avant 150 L; Cytiva). Equal amounts (8 µg per well) of purified etanercept were analyzed on Novex™ pH 3–7 IEF Protein Gels (Thermo Fisher Scientific) as described previously (Lee et al. 2024).

N-glycan structure analysis of etanercept

The N-glycans of etanercept were released, labeled with 2-aminobenzamide (2-AB), and analyzed by UHPLC following a previously described protocol (Lee et al. 2024), with minor modifications detailed below. The labeled N-glycans were injected directly into an AdvanceBio Glycan Mapping column (1.8 μm, 2.1 mm × 150 mm; Agilent Technologies, Santa Clara, CA, USA) connected to a UHPLC system (1290 Infinity Ⅱ Bio LC system; Agilent Technologies). They were monitored using a fluorescence detector at excitation and emission wavelengths of 260 and 430 nm, respectively.

O-glycan structure analysis of etanercept

To release the O-glycan moieties, purified etanercept was dialyzed in 0.1% trifluoroacetic acid solution and dried completely. The O-glycans were released using a Ludger Liberate™ Orela Glycan Release Kit (Ludger Ltd, Oxfordshire, UK) following the manufacturer’s instructions. After labeling the released free O-glycans with 2-AB, they were separated using an AdvanceBio Glycan Mapping column (1.8 μm, 2.1 mm × 150 mm; Agilent Technologies) connected to a UHPLC system (1290 Infinity Ⅱ Bio LC system; Agilent Technologies). The labeled O-glycans were detected using a fluorescence detector at excitation and emission wavelengths of 260 and 430 nm, respectively.

Sialic acids content analysis of etanercept

Sialic acids in purified etanercept were profiled and quantitated using an AdvanceBio Sialic Acid Profiling and Quantitation Kit (Agilent Technologies) with a UHPLC system (1290 Infinity Ⅱ Bio LC system; Agilent Technologies), following the previously described method (Lee et al. 2024). The detected sialic acids were classified as N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) based on kit standards.

Quantitation of the intracellular CMP-SA concentration

The intracellular nucleotide sugars extracted from the cells were analyzed as described previously (Lee et al. 2024). Briefly, 3.0 × 106 cells were collected and resuspended in ice-cold 500 mM perchloric acid (Sigma-Aldrich) and neutralized with ice-cold 2.5 M KOH (Sigma-Aldrich) in 1.1 M K2HPO4 (Sigma-Aldrich). Intracellular nucleotide sugars were then quantified by ion-pair reversed-phase HPLC, which were detected at 254 nm using a Waters ACQUITY Premier HSS T3 column with a VanGuard FIT (1.8 μm, 2.1 mm × 150 mm; Waters, Milford, MA, USA) connected to a UHPLC system (1290 Infinity Ⅱ Bio LC system; Agilent Technologies). The standard chemical CMP-SA was used for peak identification and integration of the nucleotide sugars. The integrated peak area of the standard chemical was calculated using OpenLab CDS ChemStation software (Agilent Technologies) following UHPLC analysis.

Statistical analysis

The results are expressed as the mean ± standard deviation. The data were analyzed using a two-tailed student’s t-test where appropriate. Differences between the means were considered significant at p ≤ 0.05.

Results

Effects of sialylated oligosaccharides on cell growth and etanercept production

To determine the effect of sialylated oligosaccharides on cell growth and etanercept production, two rCHO cells producing etanercept were cultured with six enzymatically synthesized oligosaccharides and lactose. These molecules consisted of oligosaccharides, such as lactose, lacto-N-tetraose, and lacto-N-neo-tetraose, with and without sialic acid residues attached to the terminal or subterminal galactose by α2,3 and/or α2,6 linkages (Fig. 1). Cells without oligosaccharides were also cultivated as controls.

Fig. 1.

Fig. 1

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Structures of enzymatically-synthesized sialylated and non-sialylated oligosaccharides

Figure 2 shows profiles of viable cell concentration, cell viability, and etanercept concentration for the two rCHO cells cultured with or without enzymatically synthesized oligosaccharides. In both rCHO cells, oligosaccharide supplementation did not affect cell growth and etanercept production compared with the control cultures. The glucose, lactate, glutamine, and ammonia concentrations during culture shown in Fig. 2 were measured and showed similar trends in both rCHO cells regardless of the addition of oligosaccharides (data not shown). The results indicate that the addition of enzymatically synthesized oligosaccharides does not significantly affect overall cell growth, etanercept production, or metabolism in rCHO cell cultures.

Fig. 2.

Fig. 2

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Profiles of cell growth, viability, and etanercept production in the absence and presence of six enzymatically synthesized oligosaccharides and lactose in two rCHO cell lines. a CHO-ETN-L clone and b CHO-ETN-H clone. Without oligosaccharide (open circle), with 3′-SL (closed triangle), with 6′-SL (closed inverted triangle), with LSTa (closed square), with DSLNnT (closed diamond), with lactose (open triangle), with LNT (open square), and with LNnT (open diamond). All oligosaccharides were supplemented at a final concentration of 10 mM on day 3 of culture. The arrow indicates the time of oligosaccharide addition to the culture media. Error bars represent standard deviations calculated from triplicate experiments

Effects of sialylated oligosaccharides on the isoform distribution of etanercept

To determine the effect of sialylated oligosaccharides on the isoform distribution of etanercept in two rCHO cells with relatively low (CHO-ETN-L clone) and high (CHO-ETN-H clone) levels of sialylation, etanercept produced on day 6 of culture in Fig. 2 was purified by protein A chromatography. The purified etanercept proteins were analyzed using Coomassie Blue-stained IEF gels. To avoid the effects of sialidase released from dead cells, culture supernatants with viability greater than 90% were harvested during culture.

Figure 3 shows the isoform distribution of etanercept produced from CHO-ETN-L and CHO-ETN-H clones with and without four sialylated oligosaccharides and three non-sialylated oligosaccharides on day 6. Consistent with the results in Fig. S1, etanercept produced by the CHO-ETN-H clone showed a higher proportion of acidic isoforms below pH 5.3/5.2 compared with that of the CHO-ETN-L clone in the absence of oligosaccharide supplementation. Compared with the control cultures, the proportion of acidic etanercept isoforms below pH 5.3/5.2 in cultures with 3′-SL, 6′-SL, LSTa, and DSLNnT was significantly increased in both clones. In contrast, in cultures supplemented with lactose, LNT, and LNnT, which do not contain sialic acid residues, the proportion of acidic isoforms was similar in both clones compared with cultures without oligosaccharides. The results indicate that the addition of sialylated oligosaccharides present with sialic acid residues is efficient for increasing the sialylation of etanercept produced by rCHO cells.

Fig. 3.

Fig. 3

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Isoelectric focusing (IEF) gel analysis of purified etanercept produced in a the CHO-ETN-L clone and b the CHO-ETN-H clone. Samples were collected on day 6 of the cultures shown in Fig. 2 and purified using protein A affinity chromatography. IEF gel analysis was validated from triplicate experiments

Effects of sialylated oligosaccharides on the N-glycan profiles of etanercept

To determine the effects of sialylated oligosaccharide supplementation on the N-glycan profiles of etanercept in two rCHO cells, the N-glycan of etanercept was analyzed by hydrophilic interaction chromatography. The major N-glycans released from purified etanercept by PNGase F treatment were classified into eight groups (G1F, G1S1F, G2, G2F, G2S1, G2S1F, G2S2, and G2S2F) based on their structure.

Figure 4 shows the proportions of the major N-glycans of etanercept produced from rCHO cells supplemented with six enzymatically synthesized oligosaccharides and lactose. The proportions were calculated by integrating the area of each group following UHPLC analysis. Several glycoforms of etanercept exhibited similar trends, regardless of whether the oligosaccharides were added. However, in both clones, the proportions of the two glycoforms, G1S1F and G2, were decreased in cultures supplemented with sialylated oligosaccharides (3′-SL, 6′-SL, LSTa, and DSLNnT), whereas the proportions of the di-sialylated glycoforms, G2S2 and G2S2F, were increased significantly. For the CHO-ETN-L clone, the proportion of the G2S2F glycoform was increased the most following the addition of 3′-SL, 6′-SL, LSTa, and DSLNnT to 11.0% ± 0.1%, 9.5% ± 0.2%, 11.3% ± 0.1%, and 11.7% ± 0.1%, respectively, compared with the control culture (5.2% ± 0.1%) (Fig. 4a). A similar result was observed for the CHO-ETN-H clone. Compared with the control culture (7.6% ± 0.1%), supplementation with sialylated oligosaccharides increased the proportion of the G2S2F glycoform to 13.7% ± 0.3%, 12.1% ± 0.1%, 12.8% ± 0.1%, and 13.6% ± 0.1% by adding 3′-SL, 6′-SL, LSTa, and DSLNnT, respectively (Fig. 4b).

Fig. 4.

Fig. 4

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Hydrophilic interaction UHPC analysis of N-glycan obtained from purified etanercept in the a CHO-ETN-L clone and b CHO-ETN-H clone on day 6. The relative abundance of the N-glycan profiles obtained from cultures without oligosaccharide (black), with 3′-SL (white), with 6′-SL (light gray), with LSTa (gray), with DSLNnT (dark gray), with lactose (white striped), with LNT (gray striped), and with LNnT (dark gray striped). Error bars represent standard deviations calculated from triplicate experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Figure 5 shows the proportion of di-sialylated N-glycans (G2S2 and G2S2F) in the total N-glycans with and without oligosaccharides on day 6 in both clones. For the CHO-ETN-L clone, the proportion of di-sialylated N-glycan in the control culture was 7.2% ± 0.1%, however, following the addition of 3′-SL, 6′-SL, LSTa, and DSLNnT, the proportion was significantly increased to 14.8% ± 0.1%, 12.8% ± 0.2%, 15.2% ± 0.1%, and 16.1% ± 0.1%, respectively, which was up to 2.2-fold compared with the control culture (p ≤ 0.001) (Fig. 5a). A similar trend was observed for the CHO-ETN-H clone. Compared with the control culture (10.8% ± 0.1%), the addition of 3′-SL, 6′-SL, LSTa, and DSLNnT increased the proportion of di-sialylated N-glycan to 18.8% ± 0.5%, 16.9% ± 0.1%, 17.7% ± 0.2%, 18.9% ± 0.2%, respectively, which was up to 1.8-fold (p ≤ 0.01) (Fig. 5b). These results indicate that supplementation with sialylated oligosaccharides significantly increases the proportion of di-sialylated N-glycans of etanercept produced by rCHO cells.

Fig. 5.

Fig. 5

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The proportion of di-sialylated N-glycans (G2S2 and G2S2F) in the total N-glycans of etanercept with and without six enzymatically-synthesized oligosaccharides and lactose in two rCHO cells, a CHO-ETN-L clone and b CHO-ETN-H clone. Without oligosaccharide (black), with 3′-SL (white), with 6′-SL (light gray), with LSTa (gray), with DSLNnT (dark gray), with lactose (white striped), with LNT (gray striped), and with LNnT (dark gray striped). Error bars represent standard deviations calculated from triplicate experiments. **p ≤ 0.01, ***p ≤ 0.001

Effects of sialylated oligosaccharides on the O-glycan profiles of etanercept

Etanercept contains 13 potential O-glycosylation sites in a single chain, which are almost exclusively a core 1 structure and may contain a sialic acid residue. To determine the effects of sialylated oligosaccharide supplementation on the O-glycosylation of etanercept, the O-glycans of etanercept were released by β-elimination reaction and analyzed by hydrophilic interaction chromatography. Unlike N-glycosylation in which several forms of glycoform are present, the O-glycan of etanercept predominantly consists of only three forms (neutral, mono-, and disialylated), of which the mono-sialylated glycoform accounts for a large proportion (Houel et al. 2014).

Figure 6 shows the proportion of three O-glycoforms of etanercept in cultures with and without oligosaccharides on day 6 in two rCHO cells. Similar to the previous studies (Cho et al. 2016; Houel et al. 2014), the proportion of mono-sialylated O-glycan in the control culture was close to 90% of the total for both clones. The addition of seven oligosaccharides did not affect the total proportion of sialylated O-glycans; however, some sialylated oligosaccharides had a significant effect on the proportion of mono- and di-sialylated O-glycans. In particular, LSTa supplementation significantly increased the proportion of di-sialylated O-glycans in the CHO-ETN-L clone by 9.7-fold to 38.2% ± 0.8% compared with the control cultures (3.9% ± 0.2%) (Fig. 6a). In the CHO-ETN-H clone, the proportion of di-sialylated O-glycans increased to 25.9% ± 1.2% and 31.9% ± 1.1% by the addition of LSTa and DSLNnT, respectively (Fig. 6b). Thus, some sialylated oligosaccharides increase the di-sialylation of the O-glycan in etanercept produced in rCHO cells.

Fig. 6.

Fig. 6

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Hydrophilic interaction UHPC analysis of O-glycan obtained from purified etanercept in the a CHO-ETN-L clone and b CHO-ETN-H clone on day 6. The relative abundance of the O-glycan profiles obtained from cultures without oligosaccharide (black), with 3′-SL (white), with 6′-SL (light gray), with LSTa (gray), with DSLNnT (dark gray), with lactose (white striped), with LNT (gray striped), and with LNnT (dark gray striped). Error bars represent standard deviations calculated from triplicate experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001

Effects of sialylated oligosaccharides on the sialic acid content of etanercept

Non-human mammalian cells, including rCHO cells, express glycoproteins that contain sialic acid exhibiting Neu5Ac and Neu5Gc forms. Neu5Gc causes immunogenicity issues as it is considered a non-human epitope in the body (Borys et al. 2010). Therefore, we determined the effect of enzymatically synthesized oligosaccharides on the sialic acid content of the Fc-fusion glycoprotein from etanercept-producing rCHO cells using UHPLC.

Figure 7 shows the relative sialic acid content of etanercept in cultures on day 6 in the absence and presence of enzymatically synthesized oligosaccharides. The values of each oligosaccharide were normalized to the control cultures. Consistent with the N- and O-glycan structural analysis, the total sialic acid content of etanercept was increased only in cultures supplemented with sialylated oligosaccharides (3′-SL, 6′-SL, LSTa, and DSLNnT). In the CHO-ETN-L clone, the addition of 3′-SL, 6′-SL, LSTa, and DSLNnT increased Neu5Ac content by 1.3-, 1.2-, 1.6-, and 1.4-fold, respectively, compared with the control (p ≤ 0.05). In contrast, Neu5Gc content was extremely low and was not affected by the addition of sialylated oligosaccharides. Similar results were observed in the CHO-ETN-H clone, in which Neu5Ac content was increased by 1.2-, 1.3-, 1.3-, and 1.3-fold by the four sialylated oligosaccharides (3′-SL, 6′-SL, LSTa, and DSLNnT), respectively (p ≤ 0.05). Compared with the CHO-ETN-L clone, Neu5Gc content was slightly higher, but not affected by the sialylated oligosaccharides.

Fig. 7.

Fig. 7

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The relative sialic acid content of etanercept in the a the CHO-ETN-L clone and b the CHO-ETN-H clone on day 6. Without oligosaccharide (black), with 3′-SL (white), with 6′-SL (light gray), with LSTa (gray), with DSLNnT (dark gray), with lactose (white striped), with LNT (gray striped), and with LNnT (dark gray striped). The relative content of the two sialic acids, Neu5Ac and Neu5Gc, was normalized to the Neu5Ac content of etanercept on day 6 in the control cultures. Error bars represent standard deviations calculated from triplicate experiments. *p ≤ 0.05

Effects of sialylated oligosaccharides on the intracellular CMP-SA concentration

During sialylation, a sufficient amount of the donor nucleotide sugar, CMP-SA, is required to transfer a sialic acid residue to the glycan (Lawrence et al. 2001). In our previous study, the addition of sialyllactose significantly increased the intracellular levels of CMP-SA, which also resulted in the enhanced sialylation of the glycoproteins (Lee et al. 2024). To determine whether enhanced sialylation of etanercept by sialylated oligosaccharides was driven by the increased CMP-SA concentration, we measured the concentration of CMP-SA in cultures with and without oligosaccharides using an ion-pair reversed-phase HPLC method.

Figure 8 shows the relative fold-change of intracellular CMP-SA in the absence and presence of enzymatically synthesized oligosaccharides on day 6 in rCHO cells. The values for the effectiveness of each oligosaccharide were normalized to the control cultures. In the CHO-ETN-L clone, only four sialylated oligosaccharides (3′-SL, 6′-SL, LSTa, and DSLNnT) increased the concentration of intracellular CMP-SA, and non-sialylated oligosaccharides were not effective at increasing CMP-SA concentration (Fig. 8a). The addition of 3′-SL, 6′-SL, LSTa, and DSLNnT significantly increased the concentration of CMP-SA by 13.9-, 16.4-, 14.3-, and 45.8-fold, respectively, compared with that in the control culture (p ≤ 0.01). A similar trend was observed in the CHO-ETN-H clone, but to a lesser degree (Fig. 8b). The concentration of CMP-SA was increased by 7.3-, 8.4-, 11.2-, and 22.5-fold following the addition of 3′-SL, 6′-SL, LSTa, and DSLNnT, respectively (p ≤ 0.05). For both clones, the concentration of CMP-SA was significantly increased in DSLNnT, which had more sialic acid residues on the oligosaccharide. Unlike other sialylated oligosaccharides (3′-SL, 6′-SL, and LSTa), which have one sialic acid residue, DSLNnT contains two sialic acids in an α2,6-linkage (Fig. 1). The results suggest that the sialic acid residues of the sialylated oligosaccharides may be used as direct precursors for intracellular CMP-SA synthesis.

Fig. 8.

Fig. 8

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Fold-change of intracellular CMP-SA in the a CHO-ETN-L clone and b CHO-ETN-H clone on day 6. Without oligosaccharide (black), with 3′-SL (white), with 6′-SL (light gray), with LSTa (gray), with DSLNnT (dark gray), with lactose (white striped), with LNT (gray striped), and with LNnT (dark gray striped). CMP-SA was extracted and quantified from cells harvested on day 6 using ion-pair reversed-phase HPLC. The value of each oligosaccharide was normalized to the control cultures. Error bars represent standard deviations calculated from triplicate experiments. *p ≤ 0.05, **p ≤ 0.01

Validation of the effectiveness of sialylated oligosaccharides in HEK293 cells

To further explore the potential of sialylated oligosaccharides as supplements in mammalian cell culture, their effect was examined in etanercept-producing HEK293 cells. Four sialylated oligosaccharides (3′-SL, 6′-SL, LSTa, and DSLNnT) were individually administered to the HEK293-ETN clone on day 3, and samples were taken on day 6 to assess isoform distribution and intracellular CMP-SA concentration.

Figure 9 shows the isoform distribution of etanercept, the relative fold-change of CMP-SA in the HEK293-ETN clone, and a comparison of the amount of CMP-SA between the two rCHO cells and HEK293 cells producing etanercept. In contrast to the positive effect in rCHO cells, the sialylated oligosaccharides did not increase sialylation of etanercept produced by the HEK293-ETN clone (Fig. 9a). In addition, the concentration of CMP-SA did not increase dramatically following the addition of sialylated oligosaccharides (Fig. 9b). Interestingly, the control cultures of the two rCHO cells without sialylated oligosaccharides contained relatively low levels of CMP-SA (0.95 pmol/105 cells for the CHO-ETN-L clone and 3.08 pmol/105 cells for the CHO-ETN-H clone), whereas the control cultures of the HEK293-ETN clone contained significantly higher levels of CMP-SA (64.6 pmol/105 cells) (Fig. 9c). Taken together, sialylated oligosaccharides are presumed to be unsuitable for enhancing sialylation in HEK293 cells based on the evidence that HEK293 cells already have sufficient basal levels of CMP-SA to synthesize sialylated glycans.

Fig. 9.

Fig. 9

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Effect of four sialylated oligosaccharides in HEK293 cells producing etanercept. a IEF gel analysis of purified etanercept produced in the HEK293-ETN clone on day 6. b Fold-change of intracellular CMP-SA in HEK293 cells in the absence and presence of four sialylated oligosaccharides (3′-SL, 6′-SL, LSTa, and DSLNnT). The value of each sialylated oligosaccharide was normalized to the control culture. c Comparison of the total amount of intracellular CMP-SA in the CHO-ETN-L clone, CHO-ETN-H clone, and HEK293-ETN clone in cultures without the addition of oligosaccharide. Error bars represent standard deviations calculated from triplicate experiments. **p ≤ 0.01

Discussion

Ensuring the safety and efficacy of recombinant therapeutic proteins produced by mammalian cell-based bioprocesses requires close monitoring of the desired glycosylation profile. Regulatory agencies require strict control of glycan profiles during the manufacturing process to ensure the quality of therapeutic proteins as glycosylation is a critical quality attribute that affects therapeutic efficacy, including pharmacokinetics, immunogenicity, stability, and in vivo half-life (Liu 2015). Sialylation, the final step in N- and O-glycosylation, is particularly important in determining in vivo clearance. This effect is more pronounced for Fc-fusion glycoproteins, in which a higher sialic acid content results in slower clearance compared with the monoclonal antibody (Flesher et al. 1995; Liu et al. 2013). Therefore, various strategies to enhance the sialylation of recombinant proteins produced by rCHO cells have been tried, such as supplementation with nucleotide sugar precursors (Blondeel and Aucoin 2018). In particular, ManNAc has been used as a supplement for decades because it directly affects the sialylation of glycoproteins. In our recent study, enzymatically synthesized sialyllactose was confirmed as a potential supplement to enhance glycoprotein sialylation (Lee et al. 2024). In the present study, we examined the effect of several enzymatically synthesized sialylated oligosaccharides as novel supplements to enhance the sialylation of etanercept in rCHO cell culture.

Etanercept is a therapeutic Fc-fusion protein used to treat inflammatory diseases, such as rheumatoid arthritis, by blocking tumor necrosis factor (TNF) activity. It consists of the extracellular domain of the TNF receptor linked to the Fc region of IgG1 and has a total of 3 N- and 13 O-glycosylation sites (Montacir et al. 2018). In contrast to therapeutic monoclonal antibody, in which sialylation does not significantly increase the in vivo half-life, enhancing the sialylation of etanercept has significant therapeutic advantages, particularly by extending serum half-life and reducing clearance from the body. In some physicochemical and functional studies, the presence of sialic acid in the N-glycans of etanercept increases the in vivo half-life, thereby slowing down its clearance from the serum (Montacir et al. 2018). Furthermore, sialic acids can protect etanercept from oxidative damage and maintain its biological activity, such as complement-dependent cytotoxicity (CDC) (Martínez et al. 2020). The removal of sialic acid resulted in an increased rate of oxidation and a decrease in CDC activity, indicating that sialic acid in N-glycans is essential for maintaining stability and functionality.

In the present study, the addition of sialylated oligosaccharides successfully increased the sialylation of N-glycan in etanercept produced in two rCHO cells with relatively low and high levels of sialylation. In particular, the proportion of the non-sialylated (G2) and mono-sialylated glycoforms (G1S1F) of N-glycan was significantly decreased, whereas the proportion of di-sialylated glycoforms (G2S2 and G2S2F) was significantly increased in both clones (Figs. 4 and 5). The results suggest that the incomplete sialylated glycoforms of the bi-antennary N-glycans of etanercept mature to complete sialylated N-glycans through the addition of sialylated oligosaccharides. Interestingly, in the two rCHO cell lines with low and high levels of sialylation, the degree of increase to complete sialylated N-glycans by the addition of sialylated oligosaccharides was similar (Figs. 4 and S2). Furthermore, among the sialylated oligosaccharides analyzed, the oligosaccharide with the optimal effect on the sialylation of glycoproteins differed between the two rCHO cells, with LSTa being most effective in the CHO-ETN-L clone and DSLNnT in the CHO-ETN-H clone (Fig. 7). These findings may reflect intrinsic differences in the glycosylation machinery or substrate preferences of each clone and suggest the importance of having a diverse set of supplement candidates, as suggested in this study, to achieve optimal sialylation enhancement.

Compared with N-glycosylation, examining the effect of O-glycosylation on the efficacy and functionality of therapeutic proteins remains limited (Thompson and Wakarchuk 2022). Nevertheless, a few studies have reported that the O-glycan of some glycoproteins may exert a beneficial effect on therapeutic efficacy. The specific O-glycoforms of etanercept produced by rCHO cells engineered to express various O-glycovariants exhibit enhanced biological activity (Biel et al. 2022). This study showed that increased sialylation of O-glycan resulted in increased binding affinity for TNF and improved safety by suppressing the immune response. Therefore, studies optimizing culture conditions and evaluating culture supplements that modulate the sialylation of O-glycan are valuable, but few have been reported to date. The O-glycans present in etanercept are predominantly in a mono-sialylated form, and even in the present study, mono-sialylated O-glycan comprised approximately 90% of the total O-glycans (Cho et al. 2016; Houel et al. 2014). Interestingly, supplementation of the sialylated oligosaccharides, LSTa and DSLNnT, significantly increased the proportion of di-sialylated O-glycan in rCHO cells, whereas the optimal effect varied among clones (Figs. 6 and Fig. S3). Based on this result, sialylated oligosaccharides can be expected to be a novel supplement to regulate the sialylation of O-glycan in rCHO cell culture, although further studies of other therapeutic proteins or cell lines are needed.

The increase in sialic acid content of etanercept was likely the result of an increase in the concentration of CMP-SA, a key nucleotide sugar in the sialic acid biosynthetic pathway (Fig. 8). Because activated nucleotide sugars are used as donor substrates for glycosyltransferase, their availability and abundance may influence the flux of glycosyltransferase reactions, thus regulating N-glycan synthesis (Burleigh et al. 2011). Notably, the concentration of CMP-SA increased in proportion to the number of sialic acids attached to the oligosaccharides in both rCHO cells (Fig. 8). DSLNnT has two sialic acid residues and exhibits a significantly greater increase in CMP-SA compared with other sialylated oligosaccharides (3'-SL, 6'-SL, and LSTa), which have one sialic acid residue (Figs. 1 and 8). However, the larger increase in CMP-SA concentration by DSLNnT supplementation did not correspond to an increase in the sialic acid content of etanercept (Fig. 7). Moreover, oligosaccharides without sialic acid (Lactose, LNT, and LNnT) were not effective at increasing the concentration of other intracellular nucleotide sugars, including CMP-SA, UDP-galactose, UDP-glucose, UDP-N-acetylglucosamine, and GDP-fucose (Fig. S4). These results suggest that the sialic acid residues of sialylated oligosaccharides act as precursors in the CMP-SA synthesis pathway, and rCHO cells may lack the relevant metabolic systems to utilize non-sialylated oligosaccharides.

The precise mechanism by which sialylated oligosaccharides enhance the sialylation of etanercept has not been elucidated. In previous studies, sialic acid residues on oligosaccharides are hydrolyzed by sialidase 1 (Neu 1) in the lysosome and transported to the cytosol, where they may be used as intermediates in the sialic acid biosynthetic pathway (Frisch and Neufeld 1979; Sprenger and Duncan 2012). Based on these findings, we attempted to determine the mechanism of action of sialylated oligosaccharides by treating them with 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), a sialidase inhibitor. However, despite this treatment, the proportion of di-sialylated N-glycans in etanercept and the concentration of intracellular CMP-SA were similar in the presence and absence of DANA (data not shown). Indeed, it is unclear whether DANA was administered at a sufficient concentration and at the optimal time to inhibit only Neu 1 in the two etanercept-producing clones used in this study. Therefore, it is necessary to clarify the mechanism of action of sialylated oligosaccharides using methods that can selectively inhibit only Neu 1, such as a genetic engineering strategy.

The glycosylation profile of recombinant proteins can vary significantly depending on the host cell line used for production because of the different host systems involved in the glycosylation pathway (Goh and Ng 2018). HEK293 cells, along with rCHO cells, are one of the most widely used mammalian cells for the production of recombinant proteins, particularly in transient gene expression systems (Park et al. 2021). It was reported that HEK293 cells tend to produce glycoproteins with more complex glycan structures (Croset et al. 2012). Unexpectedly, the addition of sialylated oligosaccharides did not significantly increase the sialylation of etanercept in HEK293 cells, unlike rCHO cells, and only slightly increased the concentration of CMP-SA (Figs. 9a, 9b, and Fig. S5). Furthermore, we reconfirmed the same results in other clones from etanercept-producing HEK293 cells (data not shown). From the evidence that etanercept-producing HEK293 cells contained significantly higher levels of CMP-SA compared with both etanercept-producing rCHO cells (Fig. 9c), we predicted that HEK293 cells may already possess sufficient basal levels of CMP-SA to naturally produce sialylated glycans. In order to generalize this hypothesis, we need to verify it using various HEK293 cells that produce Fc-fusion glycoprotein containing tetra-antennary N-glycan, and also investigated the cause of high intracellular CMP-SA concentration only in HEK293 cells.

In conclusion, we demonstrated that several enzymatically-synthesized sialylated oligosaccharides increase the proportion of di-sialylated N- and O-glycans in etanercept produced in rCHO cells with a significantly increased concentration of CMP-SA, a key nucleotide sugar in the sialylation process.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 607 KB) (606.6KB, docx)

Author contributions

THK, HML, JY and YGK designed the research and wrote the manuscript. JY and YGK supervised the project. THK and HML conducted the main experiments. SHR conducted the cell line development. HSK contributed to the protein purification. SJK and MJK contributed to the protein quality analysis. All authors contributed to the writing of the manuscript and read and approved it.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program (KGM1262511) and the Technology Innovation Program funded by the Ministry of Trade, Industry & Energy (Project Number: RS-2024–00438191).

Data availability

The data that support for the findings of this study are all contained in the manuscript.

Declarations

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Tae-Ho Kim and Hoon-Min Lee contributed equally to this work.

Contributor Information

Jungmok You, Email: jmyou@khu.ac.kr.

Yeon-Gu Kim, Email: ygkim@kribb.re.kr.

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Supplementary Materials

Supplementary file1 (DOCX 607 KB) (606.6KB, docx)

Data Availability Statement

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