Chiang Mai Journal of Science

Impact of Copper and Melezitose Supplementation on Adalimumab Production and N-linked Glycosylation in Chinese Hamster Ovary Cell Culture

1. INTRODUCTION

Therapeutic monoclonal antibodies (mAbs) are typically produced in Chinese hamster ovary (CHO) cells, which generate human-like glycosylation patterns [1, 2]. Adalimumab (Humira; AbbVie) is a monoclonal antibody (mAb) targeting tumor necrosis factor-alpha (TNF-α) [3]. It is used to treat rheumatoid arthritis and other autoimmune disorders [4]. The Fc region of adalimumab contains an N-linked glycan at Asn-297 within the CH2 domain on each heavy chain [5]. In addition to TNF-α neutralization, adalimumab mediates ADCC and CDC through binding of its Fc region to Fcγ receptors and complement protein C1q, respectively [5]. This cytotoxic mechanism may contribute to adalimumab's therapeutic effect in inflammatory bowel disease [6].

Glycosylation is an important product quality attribute for recombinant antibodies because it directly affects their therapeutic efficacy and pharmacokinetics, such as immune effector functions, immunogenicity, in vivo clearance, and protein stability [7-10]. For example, high-mannose glycans increase the serum clearance of antibodies [11]. Higher levels of afucosylation enhance antibody-dependent cell-mediated cytotoxicity (ADCC) by increasing FcγRIIIa binding [12, 13]. Increased galactosylation can improve complement-dependent cytotoxicity (CDC) [14], and sialylation can affect circulatory half-life [15]. Therefore, protein glycosylation requires tight regulation in biopharmaceutical manufacture, particularly for ensuring biosimilar approval.

To gain approval, biosimilar developers must demonstrate that their product is highly similar to the reference product, with no clinically meaningful differences in safety, efficacy, or quality, in accordance with EMA and FDA regulatory guidelines [16, 17]. For adalimumab, investigators compared a number of biosimilars with Humira, characterizing various product quality attributes. Liu et al. compared ABP 501 (Amgen's adalimumab biosimilar) with adalimumab (US) and adalimumab (EU). All products showed similar glycan profiles, with major glycans present and only minor differences for low abundance glycans. Galactosylation, high mannose, and total afucosylation levels were comparable across products, consistent with the similar CDC and ADCC activities observed. Sialylated glycans were slightly higher in ABP 501 than in Humira, though levels remained at or below 1% and did not affect ADCC activity [18]. Jiang et al. found Humira and adalimumab-aqvh (FDA-approved biosimilar) comparable for most glycan categories, including high mannose, afucose, and total afucose. Differences in terminal galactose and sialic acid did not translate into functional differences in C1q binding, ADCC, or CDC activities [5]. Kwon et al. showed similar overall N-glycan profiles between Humira and LBAL (LG Chem's Humira® biosimilar), with neutral complex-type biantennary glycans predominating and afucosylated and high mannose glycans present at low abundance. LBAL had similar galactosylation but slightly higher afucosylation and sialylation, with lower high mannose levels. Binding activities for C1q, FcγRs, and FcRn, as well as CDC activity, were similar. Although LBAL showed slightly lower ADCC potency in a reporter bioassay, no differences were observed using physiologically relevant PBMCs [19].

While there are many ways to control and manipulate glycosylation patterns of therapeutic antibodies, supplementing the cell culture medium with additives provides a simple and efficient method to achieve or approach a desired glycosylation pattern [20-22]. Copper is an essential metal that plays a vital role in cellular homeostasis [23, 24]. Several studies have shown that copper, either alone or in combination with other trace elements or factors, can influence the glycosylation profile of recombinant proteins. Yang and coworkers reported the effects of copper (II) sulfate (CuSO₄) on N-linked glycosylation in fed-batch shake-flask cultures of immunoadhesin-expressing CHO cells. In their study, CuSO₄ was added either through the feed medium, reaching a final concentration of 0.2 mM on Day 14, or as bolus additions of 0.2 mM or 0.5 mM on Day 6. All CuSO₄ treatments led to reduced afucosylation, reduced galactosylation, and increased N-glycan charge relative to the control, without substantially affecting cell titer [25]. Leiske et al. investigated how CuSO₄, manganese (manganese sulfate monohydrate, Mn²⁺), and pH influence CHO cell growth, productivity, and N-linked glycosylation during recombinant anti-TNFα antibody production in a perfusion bioreactor. Using a three-factor, two-level (2³) full factorial design, they assessed main effects and two-way interactions. Copper was evaluated at 10 and 100 ppb, manganese at 50 and 1000 nM, and pH at 6.85 and 7.0, with JMP used for statistical modeling. Copper and manganese concentrations did not affect cell growth or productivity. pH significantly affected high-mannose glycans (Man5 and Man7), with higher pH increasing their abundance. Modeling indicated that higher manganese levels increased β-galactosylated species (A1G1F, A2G1F, A2G2F, and corresponding afucosylated forms). Elevated pH also enhanced β-galactosylation, though less strongly than manganese, while copper had no notable impact. Afucosylation (A1G0, A2G0, A1G1, A2G1, A2G2) increased with higher copper and manganese concentrations, with the highest levels observed when both metals and pH were elevated [26]. Popp and colleagues investigated how iron, copper, zinc, and manganese influence cell growth and mAb glycosylation using recombinant CHO-K1 cell lines expressing a monoclonal antibody in controlled 2-L Quad bioreactors. They first used DoE approaches to determine optimal ratios and target concentrations of these metals, running all DoE fed-batch studies in shaker or robotic cultivation systems. The selected metal concentrations and ratios were then tested in bioreactor fed-batch runs to promote biomass generation, increase glycoprotein maturation, or enhance production of immature non-fucosylated glycoproteins [27].

Additives that fine-tune glycosylation patterns without significantly impacting overall cell growth or productivity are highly valuable [9, 28, 29]. Hossler et al. demonstrated that melezitose, a trisaccharide composed of glucose and turanose, can modulate protein glycosylation without substantially affecting growth and productivity. Melezitose was evaluated at 1, 10, 25, and 50 mM in 250 mL fed-batch shaker flask cultures of a recombinant CHO cell line expressing a fully human IgG1 monoclonal antibody. Only the 50 mM condition adversely affected cell growth, reducing peak VCD relative to other conditions, although cell viability remained unaffected across all concentrations. All four conditions slightly reduced harvest titer compared to the unsupplemented control, with the 10 mM condition showing the greatest reduction. Melezitose supplementation substantially altered the glycosylation profile in a concentration-dependent manner. G1F species increased by 1%, 9%, 20%, and 28% at 1, 10, 25, and 50 mM, respectively. G2F glycans followed a similar trend, though with smaller absolute changes. These increases in galactosylation corresponded to nearly equivalent decreases in G0F and G0F-GlcNAc (referred to as -NAF glycan in this study) species. Man5 levels were reduced at all tested concentrations [30].

It has been reported that highly galactosylated mAbs demonstrate enhanced C1q binding and CDC activity in vitro, as well as increased FcγRII and FcγRIIIa binding and elevated ADCC [31-35]. Hybrid-type glycans show variable effects on effector functions. Conversion of core-fucosylated oligosaccharides from complex type to hybrid type did not affect CDC and ADCC mediated by mouse spleen cells [36], but decreased ADCC mediated by human polymorphonuclear leukocytes [37].

In this study, we modulated N-linked glycan profiles of adalimumab, a biosimilar of the anti-TNF-α antibody Humira produced in a CHO cell line. To shift the profile closer to the innovator, culture media were supplemented with copper or melezitose to increase G1F and decrease -NAF glycan levels. Effects on cell growth and productivity were also evaluated.

2. MATERIALS AND METHODS

2.1 Cell Lines, Cell Culture and Media
Development of CHO-K1 cells expressing adalimumab has been previously described [38]. We previously evaluated growth, productivity and glycosylation patterns of several clones [39]. In this study, only clone 9 was used. CHO cells were expanded in shake flasks (Thomson Optimum Growth® flasks). After thawing, cells were passaged at least twice (3–4 days per passage) at a seeding density of 0.3 × 10⁶ viable cells/mL in a 40 mL working volume of eCHO Basal Medium (Lonza) supplemented with 8 mM GlutaMAX (Thermo Fisher Scientific) and a 1:1000 dilution of anti-clumping agent (Thermo Fisher Scientific) before initiating experiments. Cultures were maintained at 37 °C, 5% CO₂, and 120 RPM in a humidified incubator prior to inoculation into fed-batch cultures.

2.2 Shake Flask Cell Culture
CHO cells were cultured in shake flasks under fed-batch conditions to evaluate the effects of copper or melezitose on cell growth and mAb glycosylation. Cultures were seeded at 0.3 × 10⁶ cells/mL in 125 mL shake flasks (Thomson Optimum Growth® flasks) with a 40 mL working volume of eCHO medium supplemented with 8 mM GlutaMAX™ and a 1:1000 dilution of anti-clumping agent. Cultures were maintained in a CO₂ incubator at 37 °C with 5% CO₂ and agitated at 120 rpm. eCHO concentrated nutrient feed was supplemented daily at 5% (v/v) of the initial culture volume starting on day 2. 1.5 mM copper(II) chloride dihydrate or 25 mM D(+)-melezitose monohydrate (Sigma-Aldrich) were supplemented into both the basal and feed media at concentrations based on previous studies [40, 41]. Glucose concentrations were measured daily and maintained at 6 g/L. Cultures were harvested when cell viability declined to approximately 60%. Fed-batch cultures were performed in duplicate.

2.3 Cell Growth and Antibody Titer Analysis
0.5 mL samples of cell culture medium were collected daily to measure cell density and viability using the trypan blue exclusion method with a Bio-Rad TC10 automated cell counter. Cell culture samples were centrifuged at 14,500 rpm for 5 minutes to remove cells, and the cell-free supernatant was stored at −20 °C until analyzed. Antibody titer was quantified using a Human IgG ELISA Antibody Pair Kit (STEMCELL Technologies) according to the manufacturer’s protocol.

2.4 Antibody Purification for N-Glycan Profile Analysis
Antibody purification methods have been described previously by Pranomphon et al. [39]. Briefly, the culture medium was centrifuged for cell separation. The antibody-containing supernatants were concentrated using Amicon® Ultra-15 centrifugal filter units with a 30 kDa molecular weight cutoff (Millipore) and were further purified using a 1-mL NAb Protein A/G spin purification kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Purified antibody samples were separated by SDS-PAGE, and the heavy-chain bands were excised and submitted for glycan analysis at the BOKU Mass Spectrometry Core Facility (Vienna, Austria). The originator Humira product (Humira; 40 mg/0.8 mL pre-filled syringe; AbbVie Inc., Chicago, IL, USA) was analyzed along with our samples.

2.5 Glycopeptide Analysis
Glycopeptide analysis was performed at the BOKU Core Facility Mass Spectrometry (CFMS), Vienna, Austria, as previously described [39]. Briefly, protein samples (10–30 µg) were processed by in-solution digestion, including reduction with dithiothreitol (Sigma Aldrich; CAS 3483-12-3), alkylation with iodoacetamide (Sigma; CAS 144-48-9), and acetone precipitation (Honeywell; CAS 67-64-1). After reconstitution in ammonium bicarbonate buffer (Sigma Aldrich; cat. no. 393215500), proteins were digested overnight with sequencing-grade trypsin at 37 °C.

The resulting peptides were separated using a C18 column (nanoEase M/Z HSS T3 Column, 100Å, 1.8 µm, 300 µm X 150 mm, Waters) with 0.1% formic acid (FA; Fluka, CAS 64-18-6) in water as the aqueous mobile phase (solvent A). Peptides were eluted using a linear gradient from 1% to 40% solvent B (80% acetonitrile (Thermo Scientific; CAS 75-05-8), 0.1% FA) over 45 minutes, followed by a 10-minute gradient from 40% to 95% B to elute larger peptides, at a constant flow rate of 6 µL/min.

Mass spectrometric detection was carried out on an Orbitrap Exploris 480 mass spectrometer (Thermo Scientific), equipped with a standard H-ESI source operated in positive ion mode using data-dependent acquisition (DDA). MS survey scans were acquired in the m/z range of 350–3200 Da, and the eight most intense precursor ions were selected for MS/MS fragmentation. Instrument calibration was performed using the Pierce FlexMix Calibration Solution (Thermo Scientific).

Putative glycopeptides were identified on MS-level based on characteristic patterns of mass shifts corresponding to N-glycan compositions, including variable numbers of N-acetylhexosamine (HexNAc), hexose, deoxyhexose, pentose, and sialic acid residues attached to a common peptide backbone. Theoretical monoisotopic masses of the glycopeptides were calculated using custom spreadsheets based on the known masses of amino acids and monosaccharides.

Manual data interpretation and glycopeptide identification were conducted using FreeStyle 1.8 (Thermo Scientific). Quantitative comparison of different glycoforms was performed based on the peak intensities of deconvoluted spectra. Glycoform annotation and data parsing were supported by the in-house developed software Glyco-parser (available at: https://github.com/lucaz88/Freestyle_parser).

2.6 Statistical Analysis
Statistical analyses were conducted using GraphPad Prism version 8.0.1 (GraphPad Software, San Diego, CA, USA).

3. RESULTS AND DISCUSSION

3.1 Impact of Copper or Melezitose in Fed-Batch Shake-Flask Cultures
To shift the N-glycosylation profile of the adalimumab produced, particularly G1F and -NAF glycans, toward that of commercial Humira, we supplemented fed-batch shake-flask cultures with copper or melezitose while also assessing effects on growth and productivity. Copper supplementation has been shown to achieve predetermined galactosylation profiles in anti-α4-integrin antibody production [42] and to control recombinant protein glycosylation [41]. Based on this literature, we selected 1.5 mM copper for the present study; a preliminary trial at 0.2 mM resulted in reduced productivity compared to control (data not shown), so this concentration was not pursued further. Reported copper concentrations in CHO cell culture media vary widely. Yuk et al. used 50-3,000 nM copper sulfate [43]; Capella Roca et al. tested copper sulfate pentahydrate at 4.0–80.1 µM [44], and Polanco et al. examined 100–200 µM copper sulfate pentahydrate [45]. However, few studies have investigated the effects of high copper concentrations (e.g., 1.5 mM) on N-linked glycosylation patterns.

25 mM melezitose was selected based on the prior work of Hossler et al. [40], who demonstrated that supplementation at 1, 10, 25, and 50 mM shifts protein glycosylation profiles, including G1F and -NAF species, in a concentration-dependent manner. Although 50 mM produced the greatest changes, it adversely affected cell growth, making 25 mM the optimal concentration in their studies.

3.1.1 Cell growth and antibody production
Cultures supplemented with 1.5 mM copper exhibited markedly lower maximum viable cell densities than control cultures (Figure 1A) and showed reduced viability during the first four production days (data not shown), likely due to copper toxicity. Viability improved after day 4, suggesting the development of tolerance to high copper levels. Copper supplementation also decreased IVCD relative to the controls (Figure 1A). Despite these effects, 1.5 mM copper increased product titer nearly 5-fold (Figure 1B) and specific productivity approximately 20-fold (Figure 1B) compared with control. The increased titer was due to the increase in cell-specific productivity; however, the role of copper in antibody production remains relatively unknown.

Compared with the originator product, the distinct glycosylation profiles observed in the copper- or melezitose-supplemented and control cultures likely reflect differences in cell culture medium, feed composition, media additives, process conditions, and expression cell line (Figure 2). These factors can modulate glycosylation by affecting (1) glycosyltransferase and nucleotide sugar transporter activity, (2) nucleotide sugar substrate availability, and (3) extracellular glycan degradation by glycosidases [48, 49].

For example, Pranomphon et al. [39] examined how manganese (Mn) and/or galactose (Gal) supplementation affects glycan profiles in two adalimumab-producing CHO-K1 clones during fed-batch culture. They used a Golgi glycosylation model to elucidate mechanisms driving elevated high mannose glycans. In both clones, Mn supplementation at 50 µM reduced Man5 species while increasing G1F and G2F species. The increase in galactosylated species is likely due to Mn serving as a metal ion cofactor that enhances B4GalT1 activity [50]. Gal supplementation increased G1F and G2F at the expense of G0F, presumably by increasing UDP-Gal availability for Golgi galactosyltransferases [29]. Combined supplementation with 50 µM Mn and 30 mM Gal increased galactosylation synergistically in both clones, yet paradoxically also elevated high mannose glycans. Computational modeling suggests that increased UDP-Gal synthesis may deplete UTP availability for UDP-GlcNAc production, leading to Man5 accumulation.

Yeo et al. compared glycan profiles of a recombinant mAb produced in CHO-K1 and CHO-DG44 cells. CHO-K1–derived mAb showed predominantly G0F glycans (~60%), with ~25% G1F and a small fraction of G2F. In contrast, CHO-DG44–derived mAb had lower G0F (20–30%), higher G1F (40–50%), and more G2F (~20%). Sialylated and high-mannose glycans were minor in both cell lines but significantly higher in CHO-DG44 [51].

Zheng et al. examined how a pH shift during the expression phase affects anti-CD52 mAb glycosylation and potency in CHO perfusion culture. In a 15-L bioreactor, they reduced pH from 7.15 ± 0.05 to 6.85 ± 0.05 at day 9. Among six major glycoforms (G0, G0F, G1, G1F, G2F, Man5), all except Man5 and G1 differed significantly between conditions. The lower pH increased galactosylation (primarily through higher G1F and G2F at the expense of G0F) and decreased fucosylation (due to elevated G0). These changes enhanced both CDC efficacy (via increased galactosylation) and ADCC efficacy (via reduced fucosylation) [52].

Wang et al. investigated the effect of ultralow pCO₂ (<25 mmHg) on mAb N-glycosylation in CHO fed-batch bioreactor cultures. Increasing headspace aeration to achieve ultralow pCO₂ reduced cell viability and mAb production but increased galactosylation from 21.36 ± 1.66% to 27.45 ± 2.13%, while fucosylation remained unchanged. They proposed that the enhanced galactosylation results from upregulated transcription of glycosyltransferases (GnT-1, B4GALT1) and nucleotide sugar transporters (SLC35A3/UDP-GlcNAc transporter, SLC35A2/UDP-Gal transporter) [53].

Copper can influence mAb glycosylation profiles, possibly through the following mechanisms. Copper is a redox-active transition metal that generates reactive oxygen species (ROS), including hydroxyl radicals, via Fenton and Haber-Weiss reactions [54, 55]. This ROS production contributes to oxidative stress during cell culture [56], and the resulting ROS accumulation and ER stress can alter glycan structure [57]. Additionally, Cu²⁺ ions serve as essential cofactors for Golgi-resident glycosylation enzymes [58]. Adjusting metal ion cofactor concentrations can also modulate glycosylation [59].

Hossler et al. [40] argued that because CHO cells lack enzymes to cleave di- and trisaccharides and rely on monosaccharide transporters for sugar uptake, the observed N-glycan shifts cannot result from direct interaction with glycosylation machinery or interference with glucose uptake. Instead, the authors propose these sugars act through cellular signaling pathways, supported by prior research showing turanose and palatinose activate MAPK signaling while sucrose and glucose do not [60]. The trend in N-glycan shifts with turanose is consistent with that observed with melezitose, though at different magnitudes, which is consistent with melezitose containing turanose as a structural component [40]. This suggests melezitose may exert its effects on N-glycan profiles through the same MAPK-mediated signaling mechanism.

In this study, supplementation with 25 mM melezitose appeared most favorable for shifting the glycosylation profile toward that of the innovator molecule. This condition increased G1F levels and reduced Man5 levels, though it had no effect on hybrid glycans (e.g., NAF). In contrast, other investigators found that supplementation with 1.5 mM copper increased undesirable high-mannose species, particularly Man5, which is associated with faster serum clearance [61, 62] and reduced ADCC and CDC activity [63, 64]. Kanda et al. confirmed that high-mannose human IgG1 had the shortest in vivo half-life in mice, followed by the hybrid type [63]. Copper supplementation at 1.5 mM and melezitose at 25 mM both slightly increased G1F glycans. Because terminal galactosylation enhances C1q binding and CDC activity [5], these shifts may improve effector function.

These findings highlight the need for an early control strategy in process development to characterize glycan composition and to optimize cell culture media components, process conditions, and other factors, thereby enabling the production of antibodies with high-quality N-glycosylation profiles and profiles that match those of an innovator mAb.

Copper and melezitose supplementation offer an economical approach to modulating glycosylation during protein production. Copper(II) chloride dihydrate costs approximately $0.37–$0.51 per gram (Sigma-Aldrich, Cat: C3279), while D-(+)-melezitose monohydrate costs $6–$8 per gram (Sigma-Aldrich, Cat: 63620). At working concentrations of 1.5 mM copper(II) chloride dihydrate (MW: 170.48 g/mol) and 25 mM melezitose (MW: 522.45 g/mol), a 15,000 L batch would require approximately 3.84 kg and 196 kg, respectively, costing $1,400–$1,960 for copper or $1.2–$1.6 million for melezitose. Bulk purchasing would further reduce these costs.

In comparison, small molecule inhibitors of glycosylation, such as kifunensine and deoxymannojirimycin (DMJ) [65], can target specific processing steps to generate desired glycoforms. However, these inhibitors cost $20,000–$200,000 per gram (Kifunensine: MP Biomedical, Cat: 0215995201; DMJ: Sigma-Aldrich, Cat: 84444-90-6). At required concentrations of 2 mg/L kifunensine and 80 mg/L DMJ [66-70], a 15,000 L production batch would consume 30 g or 1,200 g of inhibitor, respectively, costing approximately $5.6 million for kifunensine or $33.8 million for DMJ. Copper supplementation represents a cost reduction of approximately 3,000 to 4,000-fold compared to kifunensine and 17,000 to 24,000-fold compared to DMJ, while melezitose represents approximately 3.5 to 5-fold and 20 to 30-fold cost reductions compared to kifunensine and DMJ, respectively.

4. CONCLUSIONS

In this study, we modulated mAb N-linked glycosylation on adalimumab produced in CHO cells in fed-batch shake-flask cultures. Copper and melezitose were applied at specific concentrations to reduce hybrid glycans (e.g., –NAF) and increase G1F glycan levels, targeting a profile closer to the innovator mAb. We also evaluated the effect of these two modulators on cell growth and productivity. Supplementation with 1.5 mM copper or 25 mM melezitose modulated the glycosylation profile of therapeutic mAbs. Both supplements slightly increased G1F levels. Copper reduced hybrid glycans (e.g., -NAF) and increased Man5 levels, whereas melezitose decreased Man5 levels with no effect on hybrid glycans. Copper supplementation negatively affected cell growth but unexpectedly led to substantial increases in product titer and specific productivity compared with the control culture. In contrast, melezitose showed minimal effects on cell growth performance and harvest titer. Copper and melezitose may influence cell growth and glycan profiles by altering metabolic enzyme levels and glycosyltransferase activity. The addition of these supplements to cell culture media offers a relatively inexpensive and practical strategy for targeted modulation of mAb glycosylation. Further studies are required to elucidate the mechanisms by which these supplements regulate protein glycosylation and to assess the effects of copper and melezitose on additional product quality attributes, including aggregation, oxidation, and structural integrity.

AUTHOR CONTRIBUTIONS

Ranya Pranomphon: conducted the experiments, analyzed the data, drafted, edited, and reviewed the manuscript. Susan T. Sharfstein: designed the project, provided experimental guidance and financial support, and reviewed and revised the manuscript. Montarop Yamabhai: provided project conception and financial support, and reviewed the manuscript. Clemens Grünwald-Gruber: conducted the glycopeptide analysis and revised the manuscript.

CONFLICT OF INTEREST STATEMENT

The authors declare that there are no conflicts of interest.

DECLARATION OF USE OF GENERATIVE AI

During the preparation of this manuscript, ChatGPT 5.0 was used in developing the text description for the materials and methods section, based on previous publications from our laboratory. It is also understood that after using AI, the authors have reviewed and edited the manuscript accordingly and take full responsibility for its content.

FUNDING

This research has received funding support from BIOTEC, National Science and Technology Development Agency (NSTDA) [grant number P-18-50127], the NSRF via the Program Management Unit for Human Resources & Institutional Development, Research and Innovation [grant number B36G660005] and Suranaree University of Technology (www.sut.ac.th) [grant number 204194]. Ranya Pranomphon and Montarop Yamabhai were supported by the Royal Golden Jubilee (RGJ) Ph.D. Scholarship, Thailand Research Fund (TRF) [grant number PHD/0184/2558].

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