Modulation of N-Glycosylation in Bispecific Antibody Biosimilars through Combined Modulators

Modulation of N-Glycosylation in Bispecific Antibody Biosimilars through Combined Modulators | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Modulation of N-Glycosylation in Bispecific Antibody Biosimilars through Combined Modulators Xu Shengnan, Wang Pan, Liu Xiaojing, Wang Xiaofei, Mao Jiaqi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6796656/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract To ensure a high degree of similarity between biosimilars and reference drugs, it is crucial to perform comprehensive characterization and maintain rigorous control over glycosylation processes. Here,we aimed to optimize the glycosylation profile of a biosimilar of a bispecific monoclonal antibody (Bs-mAb1) to closely resemble that of the reference drug through the synergistic use of glycosylation modulators. To identify the strongest modulators and appropriate concentration ranges, we first examined the effects of different concentrations of galactose (Gal) and manganese chloride (MnCl 2 ) on the galactosylation rate in Shake Flask, as well as the influence of tris(hydroxymethyl)aminomethane (Tris) on the incorporation of mannose and fucose in 2 L Bioreactor. Importantly, the concurrentuse of Tris and galactose did not result in any interaction effects on N-glycan modifications and had no detrimental impact on cell growth, metabolism, antibody charge variants or purity. In conclusion, The concurrent use of 0.75 mM Tris and 8 mM galactose yields a glycosylation profile of Bs-mAb1 that is highly comparable to that of the reference drug, thereby providing an effective strategy for optimizing glycosylation in biosimilars. These findings provide significant insights into the regulation of glycosylation in the production of therapeutic monoclonal antibodies and may contribute to enhancing the consistency and therapeutic performance of biosimilars. Glycosylation Biosimilars Monoclonal Antibodies Additives Cell culture Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The approval and utilization of biosimilars have enhanced the accessibility of medicines and, to some extent, mitigated the rising healthcare costs. Biosimilars exhibit high similarity to their reference biologics and serve as an alternative therapeutic option for patients relying on expensive originator drugs, such as bevacizumab, rituximab, and adalimumab. In 2015, the National Medical Products Administration (NMPA) of China released its inaugural biosimilar guidelines, after which the biosimilar market in China has grown rapidly, with an anticipated market size of $ 5.6 billion by 2027[ 1 ]. In accordance with ICH Q6B guidelines and global regulatory requirements, biosimilars must demonstrate a high level of similarity to the reference product with respect to structure, function, and critical quality attributes in order to obtain regulatory approval[ 2 ]. Consequently, conducting a rigorous comparability assessment between biosimilars and their reference products is essential. Due to the proprietary and undisclosed nature of the manufacturing processes for reference drugs, establishing a production process is the initial step in biosimilar development. The production processes of biosimilars inherently differ from those of reference drugs, and variations in manufacturing methods, such as host cells, culture conditions, and purification processes, may lead to differences in the quality attributes of biotechnological products[3–5]. Biosimilars necessitate comprehensive similarity studies focusing on critical quality attributes, including primary structure (e.g., amino acid sequence and disulfide bonds), higher-order structures, post-translational modifications (such as glycosylation sites and glycans), additional modifications (e.g., oxidation, deamidation, and glycosylation), charge variants, product-related impurities (such as aggregates, fragments, and mismatched disulfide bonds), and biological activity[6–8]. Most monoclonal antibodies (mAbs) undergo one or more post-translational modifications (PTMs), which are biochemical alterations occurring after protein synthesis. These modifications, either enzymatic or non-enzymatic, affect various amino acid side chains or peptide bonds and include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and proteolysis[8, 9]. Among these, glycosylation—defined as the covalent attachment of oligosaccharides to amino acid residues such as asparagine (N-linked) or serine/threonine (O-linked) plays a particularly vital role. It significantly influences a protein's stability, conformation, receptor binding affinity, and biological activity. As such, glycosylation is widely regarded as one of the most critical PTMs in therapeutic protein development[10].Glycosylation is widely present in mammalian cells, for monoclonal antibody (mAb) therapeutics, N-linked glycosylation predominantly occurs at the conserved Asn297 residue in the Fc region[11, 12]. Common glycan structures found on antibodies include mannose, galactose, fucose, and N-acetylglucosamine. Glycosylation of the Fc (crystallizable fragment) region plays a critical role in modulating interactions with Fc gamma receptors (FcγRs), complement component C1q, and the neonatal Fc receptor (FcRn), thereby influencing antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and serum half-life[11, 13–15]. Studies have shown that immunoglobulin G (IgG) antibodies lacking Fc glycans lose their capacity to bind FcγRs and C1q, resulting in the complete loss of ADCC and CDC activity. In addition, the absence of glycosylation alters the net charge of the antibody, which can reduce its circulatory half-life [16, 17]. Fucose is typically linked to the innermost N-acetylglucosamine of N-glycans via an α-1,6 linkage. More than 90% of antibodies expressed in Chinese hamster ovary (CHO) cells exhibit core fucosylation. This modification significantly impacts ADCC, as afucosylated IgG antibodies demonstrate up to a 50-fold increase in binding affinity for FcγRIIIa and a 100-fold enhancement in ADCC, without appreciable changes in binding to FcγRI, FcγRIIa, or C1q[18]. In drug development, high-mannose glycoforms are closely monitored due to their considerable influence on pharmacokinetics (PK). Antibodies bearing mannose-rich glycans exhibit faster systemic clearance—approximately 40% higher—compared to bisected, fucosylated structures[19]. Galactose is a terminal monosaccharide commonly found on the N-glycan structures of antibodies, and its presence can affect both functional activity and pharmacokinetics. Increased galactosylation has been shown to enhance binding to complement component C1q, thereby promoting CDC. For example, galactosylation of the Fc region of rituximab correlates with improved CDC activity[20, 21]. However, galactose modification appears to have minimal influence on antibody half-life. Comparative studies of antibodies with different levels of galactosylation, such as G0F and G2F glycoforms, have demonstrated no significant differences in their in vivo pharmacokinetics[22, 23]. Glycosylation of monoclonal antibodies is a highly heterogeneous and tightly regulated post-translational modification that is critically influenced by various upstream process parameters. Among the most significant factors are the host cell line, culture medium composition, and cell culture process conditions, all of which can profoundly alter the structure and distribution of N-glycans on the Fc region of antibodies[24, 25]. The choice of expression system, particularly the host cell line, plays a central role in determining glycosylation patterns, CHO cells are widely used in therapeutic antibody production due to their human-compatible glycosylation capabilities[26]. During cell line development, genetic engineering techniques can be employed to modulate the expression of glycosyltransferases, thereby significantly altering the glycosylation patterns of antibodies. For example, knockout of the FUT8 gene in CHO cells effectively reduces core fucosylation, resulting in enhanced antibody-dependent cellular cytotoxicity (ADCC) activity[27]. Additionally, the use of endogenous internal ribosome entry sites (IRES) and polycistronic vectors to co-regulate the expression of α-mannosidase II (MANII) and β-1,4-N-acetylglucosaminyltransferase III (cGNTIII) has enabled the production of antibodies with distinct glycan profiles, leading to improved effector functions[28].Cell culture conditions, including media composition[29], the use of additives[30], pH[31], dissolved oxygen levels[32], culture temperature, and feeding strategies[33, 34], can indirectly influence glycosylation by altering cellular metabolism and the activity of glycosylation enzymes in the endoplasmic reticulum and Golgi apparatus. In recent years, advances in process analytical technologies (PAT)[35] and predictive modeling have also contributed to glycan modulation[36]. Among these approaches, the use of additives epresents one of the simplest and most effective strategies for glycan regulation. Small molecule inhibitors of mannosidases I and II, such as kifunensine and 1-deoxymannojirimycin (DMJ) are also widely used[37, 38]. However, these inhibitors degrade rapidly at room temperaturxe and are costly. Previous studies have investigated the regulation of galactosylation using various glycosylation modulators, such as galactose, uridine, manganese ions, and their mixtures[39, 40]. A growing array of culture medium additives, including metal ions, vitamins, sugars, and nucleosides, are being utilized to regulate N-glycosylation of mAbs without compromising other key quality attributes. The addition of different metal ions (Mn²⁺, Cu²⁺) and sugars (galactose, glucosamine) to the culture medium was found to significantly influence antibody glycosylation. Galactose supplementation markedly increased galactosylation levels, while Mn²⁺ and glucosamine enhanced sialylation, collectively contributing to an overall improvement in the glycan profiles[41–44]. This research focuses on optimizing the N-Glycan profile of a biosimilar monoclonal antibody to closely match that of the reference product. By first testing individual regulators at different concentrations to evaluate their effects on cell growth, expression, and N-Glycan profiles, the optimal conditions are identified. Subsequently, combinations of these regulators are applied to further refine the glycosylation pattern, aiming for high similarity with the original product. The innovative aspect of this study lies in the strategic use of multiple regulators to achieve precise glycosylation control, ensuring consistency with the reference mAb. Materials and Methods Cell Line and Reagents CHO-K1 cells (Merck), stably expressing a bispecific monoclonal antibody, were used for all experiments. The amplification medium (M1), fed-batch basal medium (M2), and feed media (FM1/FM2, mixed at a 10:1 ratio) were obtained from Cytiva. Manganese chloride (MnCl₂), galactose, and Tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma-Aldrich. Glucose was sourced from Shanghai Shenggong. Seed Expansion Cells were thawed into 125 mL shake flasks with an initial working volume of 25 mL. Cultures were maintained at 36.5°C, 5% CO₂, and 80% relative humidity with shaking at 125 rpm. Cells were passaged every three days at approximately 0.3 × 10⁶ cells/mL until the required seed volume was achieved. Shake Flask Culture Cells were inoculated into 250 mL shake flasks at an initial density of ~ 0.5 × 10⁶ cells/mL and a working volume of 50 mL. Culture conditions were as described above. On day 4, the temperature was shifted from 36.5°C to 32.0°C. The process followed a fed-batch strategy, with FM1 added at 4%, 5%, 5%, 5%, and 4% (v/v) of the initial culture volume on days 3, 6, 8, 10, and 12, respectively; FM2 was supplemented at 10% of the FM1 volume. Stock solutions of 400 mM galactose and 500 µM MnCl₂ were prepared. Galactose was added at final concentrations of 4, 8, and 10 mM, and MnCl₂ at 0.10, 0.25, and 0.40 µM, on each feeding day. Cultures were harvested on day 14. Daily samples were collected for cell density and metabolite analysis. At harvest, cultures were centrifuged at 7000 rpm for 15 minutes, and supernatants were filtered through 0.22 µm filters for subsequent purification, titer determination, and quality assessments. Bioreactor Culture Cells were inoculated into a 2 L bioreactor at an initial density of 0.5 × 10⁶ cells/mL, with a starting volume of 1.4 L. The pH was controlled at 6.9 ± 0.2, with dissolved oxygen maintained at 40%, and agitation set to 180 rpm. The feeding strategy and temperature shift followed the shake flask protocol. 500 mM Tris stock solution was prepared, and final concentrations of 0.5, 0.75 and 1.0 mM Tris were added on feeding days. 1 M galactose stock solution was also prepared, and 4, 8 and 12 mM galactose was added on feeding days. On non-feeding days, glucose concentration was maintained above 6 g/L by supplementing to 8 g/L when necessary. On feeding days, if glucose dropped below 6 g/L, it was replenished to 10 g/L. Daily samples were taken for cell growth, metabolic profiling, and osmolality measurements. The culture was harvested on day 14, followed by centrifugation at 7000 rpm for 15 minutes. Supernatants were filtered through 0.22 µm filters and used for downstream purification and quality analysis. Protein Purification, Titer, and Quality Analysis Clarified supernatants were purified by Protein A affinity chromatography using AT ProteinA Diamond Plus resin. The purified antibodies were subjected to quality assessments. Titer was quantified via Protein A affinity high-performance liquid chromatography (HPLC). Purity was evaluated using size-exclusion HPLC (SE-HPLC) with a TOSOH TSKgel G3000 SWXL column and a neutral pH mobile phase, with UV detection. Charge variants were assessed using cation-exchange HPLC (CE-HPLC) on a Thermo ProPac WCX-10 (4 × 250 mm) column, also with UV detection. For N-glycan profiling, glycans were enzymatically released using PNGase F, derivatized with 2-aminobenzamide (2-AB), and analyzed by hydrophilic interaction liquid chromatography (HILIC). Results The biosimilar Bs-mAb1 showed significant differences in glycosylation patterns—particularly in mannose, galactose, and fucose compared to the reference product. Specifically, mannose and galactose glycosylation levels were reduced by 113.7% and 90%, respectively, while fucose glycosylation was 5.0% higher. To align the glycosylation profile of the biosimilar more closely with that of the reference product, this study evaluated the effects of galactose and MnCl₂, aimed at enhancing galactosylation, and Tris, intended to modulate mannose and fucose glycosylation. Their impacts on cell growth, metabolism, antibody yield, and overall product quality were systematically assessed. Based on previous reports and internal platform data, galactose was evaluated at concentrations of 4 mM, 8 mM, and 12 mM, and MnCl₂ at 0.10 µM, 0.25 µM, and 0.40 µM. The impact on cell growth, metabolism, antibody titer, and particularly N-glycan profiles was assessed. Viable cell density and viability (Fig. 1) showed similar peak densities across groups, with cell viability maintained above 95% at harvest. Lactate metabolism (Fig. 1b) revealed that lactate levels steadily declined across all galactose concentrations. Titer data (Table 1 ) showed that the control group reached 6.1 g/L, while the galactose-treated groups maintained titers around 6.0 g/L, indicating that galactose supplementation had no adverse effects on growth, metabolism, or antibody production. Product quality analysis (Table 2 ) revealed no significant differences in main peak, acidic variants, or mispaired peaks between the galactose-treated and control groups. SE-HPLC (Table 1 ) confirmed consistent main peak content, suggesting no impact on purity. N-glycan analysis (Fig. 1) demonstrated that galactose supplementation significantly increased galactosylation without affecting mannose or fucose levels. Galactose concentrations of 4 mM, 8 mM, and 12 mM led to increases of 42.3%, 83.8%, and 85.9%, respectively, in galactosylation, exhibiting a dose-dependent effect that plateaued between 8 mM and 12 mM—consistent with previous studies. Based on these findings and the gap in galactosylation between the biosimilar and reference product, 8 mM galactose was selected for subsequent bioreactor studies. In contrast, MnCl₂ at higher concentrations negatively impacted cell proliferation. As shown in Fig. 2, the VCDEnd in the control group reached 26.0 × 10⁶ cells/mL, whereas the 0.40 µM MnCl₂ group achieved only 19.0 × 10⁶ cells/mL. Elevated MnCl₂ also led to increased lactate accumulation in the late culture phase (Fig. 2), indicating metabolic stress. Titer data (Table 1 ) showed that antibody yield in the high MnCl₂ group was approximately 30% lower than in the control. However, CE-HPLC (Table 2 ) and SE-HPLC (Table 1 ) analyses indicated that MnCl₂ had no significant effect on charge variants or aggregation. N-glycan analysis (Fig. 1f) revealed that MnCl₂ had a pronounced effect on galactosylation, with increases of 241.7%, 399.0%, and 612.7% observed at 0.10 µM, 0.25 µM, and 0.40 µM, respectively. This enhancement was dose-dependent, while mannose and fucose levels remained unchanged. Due to the excessive galactosylation and the detrimental impact on cell growth and antibody expression, MnCl₂ was not pursued in further bioreactor studies. Table 1 Effects of Galactose and MnCl 2 on Titer and purity Table 2 Effects of Galactose and MnCl 2 on antibody heterosomes Item JJ （ % ） acidic （ % ） main （ % ） basic （ % ） QQ （ % ） Control 22.7 2.4 63.6 9.7 1.6 4mM Galactose 20.8 2.5 64.5 10.8 1.4 8mM Galactose 22.1 2.5 62.2 11.6 1.6 12mM Galactose 23.8 2.6 60.9 10.9 1.7 0.10μM MnCl 2 21.9 2.5 63.4 11.1 1.2 0.25μM MnCl 2 25.3 2.7 59.3 10.8 2.0 0.40μM MnCl 2 22.3 2.6 62.7 10.8 1.5 Shake flask models have limitations due to the absence of pH and dissolved oxygen (DO) control, which can affect viable cell density (VCD) and cell viability. Additionally, studies have shown that Tris can exert cytotoxic effects at certain concentrations, especially under suboptimal pH conditions. Therefore, to more accurately evaluate the effects of Tris, experiments were conducted in a 2 L stirred-tank bioreactor, testing concentrations of 0.50 mM, 0.75 mM, and 1.00 mM. The impact on antibody glycosylation, cell growth, and metabolism was assessed. As shown in Fig. 3, different concentrations of Tris had no significant effect on cell growth, and lactate metabolism patterns remained consistent across groups. HPLC analysis (Table 3 ) indicated that the main peak content of the antibody remained around 95% across all conditions, suggesting no impact on product purity. Furthermore, CE-HPLC data (Table 4 ) confirmed that Tris did not alter the formation of acidic variants compared to the control. N-glycan analysis (Fig. 3) revealed a positive correlation between Tris concentration and mannose glycosylation, and a negative correlation with fucose glycosylation, while the impact on galactosylation was minimal. Specifically, at 0.50 mM, 0.75 mM, and 1.00 mM Tris, fucose glycosylation decreased by 2.9%, 5.6%, and 10.2%, respectively, while mannose glycosylation increased by 61.9%, 114.3%, and 204.8%. Tris primarily reduced G0F structures and increased the proportion of Man5. Among the tested concentrations, 0.75 mM Tris yielded a glycosylation profile most similar to the reference product without compromising cell growth, metabolism, or other quality attributes. Therefore, 0.75 mM Tris was selected for further study. When 0.75 mM Tris and 8 mM Galactose were added in combination, cell growth and metabolism remained unaffected (Fig. 4), except for a slight reduction in glucose consumption compared to the control. This was likely due to galactose serving as an alternative energy source, reducing glucose demand. CE-HPLC results (Table 4) confirmed that the combination of Tris and Galactose did not impact charge variant formation, and SE-HPLC data (Table 3) showed no effect on antibody purity. N-glycan analysis (Fig. 4) demonstrated that combined supplementation with Tris and Galactose resulted in a 118.2% increase in mannose glycosylation, a 78.8% increase in galactose glycosylation, and a 6.2% decrease in fucose glycosylation. Overall, this combination produced a glycosylation profile highly consistent with that of the reference product. Table 3 Effects of Tris on Titer and purity Item Titer(g/L) LW(%) HW(%) Main(%) Control 7.4 N.D. 4.5 95.5 0.5mM Tris 7.4 N.D. 4.6 95.4 0.75mM Tris 7.2 N.D. 3.8 96.2 1mM Tris 7.3 N.D. 4.4 95.6 0.75mM Tris + 8mM Galactose 7.4 N.D. 4.0 96.0 Table 4 Effects of Tris on antibody heterosomes Item JJ(%) acidic(%) main(%) basic(%) QQ(%) Control 28.1 3.7 57.3 8.5 2.3 0.5mM Tris 27.5 3.8 58.1 8.5 2.3 0.75mM Tris 27.6 3.7 58.0 8.6 2.1 1.0mM Tris 27.4 3.6 58.0 8.7 2.3 0.75m MTris + 8mM Galactose 29.1 3.9 55.9 8.8 2.2 Discussion Glycosylation is a critical quality attribute (CQA) of monoclonal antibodies, affecting their efficacy, stability, and immunogenicity. In this study, we systematically assessed the effects of galactose, MnCl₂, and Tris on the N-glycan profile, cell growth, metabolism, and product quality of a bispecific monoclonal antibody biosimilar. These additives were selected based on their established or proposed roles in modulating specific glycosylation pathways, with the goal of improving similarity to the reference product. Galactose supplementation enhanced galactosylation in a clear dose-dependent manner, with little to no impact on other glycoforms or cell culture performance. This is consistent with previous findings that exogenous galactose serves as a substrate for β-1,4-galactosyltransferase in the Golgi, increasing terminal galactose residues on N-glycans[26]. A saturation point was observed between 8 mM and 12 mM, suggesting limitations in enzyme capacity or intracellular transport[45]. Notably, galactose addition did not negatively affect cell viability, lactate metabolism, titer, or other quality attributes, making it a straightforward and effective strategy for enhancing galactosylation in biosimilar development.MnCl₂ had a more complex impact. While it significantly increased galactosylation in a dose-dependent fashion, it also impaired cell growth and productivity at higher concentrations. This is likely due to manganese’s dual role as a cofactor for glycosyltransferases, and as a cytotoxic agent when present at elevated levels[46, 47]. The accumulation of lactate and reduced viable cell density in MnCl₂ treated cultures suggest that oxidative or metabolic stress may contribute to its cytotoxicity. Given these drawbacks, MnCl₂ is less ideal for process development unless its concentration is carefully optimized. Tris, a common buffering agent, showed distinctive regulatory effects on mannose and fucose glycosylation. Increasing Tris concentrations were associated with higher mannose levels and reduced fucosylation. This may be due to Tris altering intracellular pH or influencing the activity of glycosylation enzymes such as α-mannosidase II and fucosyltransferase 8 (FUT8)[48, 49]. Although Tris can be cytotoxic under certain conditions[50], no adverse effects on cell growth, viability, or antibody quality were observed in the bioreactor system, likely due to well-controlled pH and dissolved oxygen levels.The combination of 0.75 mM Tris and 8 mM galactose produced a synergistic effect, significantly improving the glycosylation profile in terms of galactose, mannose, and fucose content, without negatively impacting cell growth, metabolism, or product quality. The absence of interference between the two additives further supports the viability of combined additive strategies in upstream process optimization. Overall, these findings provide practical insights into the targeted modulation of N-glycosylation through small-molecule additives. This strategy represents a flexible, patent-friendly, and cost-effective alternative to genetic engineering, particularly valuable in biosimilar development where close glycan similarity to the reference product is required. In addition, the experimental approach, which involves a systematic evaluation of both individual and combined additives under well-controlled bioreactor conditions, serves as a valuable reference for future glycoengineering efforts during upstream process development. Conclusion Glycosylation modifications of monoclonal antibodies and recombinant proteins are critical quality attributes that have a significant impact on drug efficacy, safety, and half-life. Key factors such as the glycosylation site, glycan type, and glycan abundance influence the biological activity, stability, and overall performance of the product. During upstream process development, glycosylation can be modulated through careful cell line selection, genetic engineering, process parameter optimization, media formulation, and feeding strategies. Although genetic modification provides precise control, process optimization and the use of regulatory additives are often preferred in industrial settings due to patent constraints and practical considerations. Among these strategies, the addition of specific small molecules represents a cost-effective and efficient approach to modulate glycosylation, particularly in biosimilar development, where close similarity to the reference product is essential.In this study, we systematically evaluated the effects of galactose, MnCl₂, Tris, and their combinations on cell growth, metabolism, antibody yield, and N-glycan profiles. Galactose and Tris, whether used individually or together, showed no adverse effects on cell performance, whereas MnCl₂ reduced both cell growth and protein expression. Notably, the combination of 8 mM galactose and 0.75 mM Tris significantly improved the glycosylation pattern. Mannose glycosylation increased by 118.2%, galactose by 78.8%, and fucose levels decreased by 6.2%, with no evidence of negative interaction between the two additives. The resulting N-glycan profile closely resembled that of the reference product.This work demonstrates a practical and effective approach for controlling glycosylation using small-molecule additives, offering a valuable reference for accelerating biosimilar development and reducing production costs. Declarations Competing Interests: The authors declare that they have no financial or non-financial interests that could be perceived as directly or indirectly influencing the work submitted for publication. References Wei, K.K., et al., Biosimilars: navigating the regulatory maze across two worlds. TRENDS IN BIOTECHNOLOGY, 2023. 41(7): p. 847-850. ICH, Test Procedures and Acceptance Criteria for Biotechnological/Biological Products, in ICH Harmonised Tripartite Guideline. 1999: . 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Jordan, Galactosylation variations in marketed therapeutic antibodies. MABS, 2012. 4(3): p. 385-391. Chang, M.M., et al., Small-molecule control of antibody N-glycosylation in engineered mammalian cells. NATURE CHEMICAL BIOLOGY, 2019. 15(7): p. 730-+. Boune, S., et al., Principles of N-Linked Glycosylation Variations of IgG-Based Therapeutics: Pharmacokinetic and Functional Considerations. ANTIBODIES, 2020. 9(2). Luo, S. and B.L. Zhang, Benchmark Glycan Profile of Therapeutic Monoclonal Antibodies Produced by Mammalian Cell Expression Systems. PHARMACEUTICAL RESEARCH, 2024. 41(1): p. 29-37. Wang, Q., et al., Application of the CRISPR/Cas9 Gene Editing Method for Modulating Antibody Fucosylation in CHO Cells. Methods in molecular biology (Clifton, N.J.), 2024. 2810: p. 249-271. Nguyen, N., et al., Optimizing effector functions of monoclonal antibodies via tailored N-glycan engineering using a dual landing pad CHO targeted integration platform. SCIENTIFIC REPORTS, 2023. 13(1). Kuwae, S., I. Miyakawa and T. Doi, Development of a chemically defined platform fed-batch culture media for monoclonal antibody-producing CHO cell lines with optimized choline content. CYTOTECHNOLOGY, 2018. 70(3): p. 939-948. Ehret, J., et al., Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells. BIOTECHNOLOGY AND BIOENGINEERING, 2019. 116(4): p. 816-830. Pacis, E., et al., Effects of Cell Culture Conditions on Antibody N-linked Glycosylation-What Affects High Mannose 5 Glycoform. BIOTECHNOLOGY AND BIOENGINEERING, 2011. 108(10): p. 2348-2358. Costa, A.R., et al., The impact of microcarrier culture optimization on the glycosylation profile of a monoclonal antibody. SPRINGERPLUS, 2013. 2. Gyorgypal, A., et al., Temporal Galactose-Manganese Feeding in Fed-Batch and Perfusion Bioreactors Modulates UDP-Galactose Pools for Enhanced mAb Glycosylation Homogeneity. BIOTECHNOLOGY AND BIOENGINEERING, 2025. Wong, D., et al., Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures. BIOTECHNOLOGY AND BIOENGINEERING, 2005. 89(2): p. 164-177. Liang, G., et al., Soft-sensor model development for CHO growth/production, intracellular metabolite, and glycan predictions. FRONTIERS IN MOLECULAR BIOSCIENCES, 2024. 11. Wang, Y., et al., Iterative learning robust optimization- with application to medium optimization of CHO cell cultivation in continuous monoclonal antibody production. JOURNAL OF PROCESS CONTROL, 2024. 137. Yu, M., et al., Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans. MABS, 2012. 4(4): p. 475-487. Levanon, S.S., et al., An efficient method to control high mannose and core fucose levels in glycosylated antibody production using deoxymannojirimycin. JOURNAL OF BIOTECHNOLOGY, 2018. 276: p. 54-62. Kildegaard, H.F., et al., Glycoprofiling Effects of Media Additives on IgG Produced by CHO Cells in Fed-Batch Bioreactors. BIOTECHNOLOGY AND BIOENGINEERING, 2016. 113(2): p. 359-366. Gramer, M.J., et al., Modulation of Antibody Galactosylation Through Feeding of Uridine, Manganese Chloride, and Galactose. BIOTECHNOLOGY AND BIOENGINEERING, 2011. 108(7): p. 1591-1602. Gangwar, N., N. Dixit and A.S. Rathore, N-Glycosylation modulators for targeted manipulation of glycosylation for monoclonal antibodies. APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 2025. 109(1). Shadrick, M., K.J. Stine and A.V. Demchenko, Expanding the scope of stereoselective ?-galactosylation using glycosyl chlorides. BIOORGANIC & MEDICINAL CHEMISTRY, 2022. 73. Prabhu, A., D. Shanmugam and M. Gadgil, Engineering nucleotide sugar synthesis pathways for independent and simultaneous modulation of N-glycan galactosylation and fucosylation in CHO cells. METABOLIC ENGINEERING, 2022. 74: p. 61-71. Gangwar, N., et al., Effect of vitamins and metal ions on productivity and charge heterogeneity of IgG1 expressed in CHO cells. BIOTECHNOLOGY JOURNAL, 2021. 16(8). Kildegaard, H.F., et al., Glycoprofiling Effects of Media Additives on IgG Produced by CHO Cells in Fed-Batch Bioreactors. BIOTECHNOLOGY AND BIOENGINEERING, 2016. 113(2): p. 359-366. Madabhushi, S.R., et al., Understanding the effect of increased cell specific productivity on galactosylation of monoclonal antibodies produced using Chinese hamster ovary cells. JOURNAL OF BIOTECHNOLOGY, 2021. 329: p. 92-103. Markert, S., et al., Traces matter: Targeted optimization ofmonoclonal antibodyN-glycosylation based on/by implementing automated high-throughput trace element screening. BIOTECHNOLOGY PROGRESS, 2020. 36(6). Kim, T.H., et al., Effect of N-Glycan Profiles on Binding Affinity of Diagnostic Antibody Produced by Hybridomas in Serum-Free Suspension. JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY, 2025. 35. Brantley, T.J., F.G. Mitchelson and S.F. Khattak, A class oflow-costalternatives to kifunensine for increasing high mannose N-linked glycosylation for monoclonal antibody production in Chinese hamster ovary cells. BIOTECHNOLOGY PROGRESS, 2021. 37(1). Feng, Y.X., et al., Tris(1,3-dichloro-2-propyl) phosphate-induced cytotoxicity and its associated mechanisms in human A549 cells. TOXICOLOGY AND INDUSTRIAL HEALTH, 2024. 40(7): p. 387-397. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6796656","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":468143469,"identity":"eb5ab013-8f62-455f-bb82-d6a22cde57a5","order_by":0,"name":"Xu 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15:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6796656/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6796656/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84236077,"identity":"ceb5a8b3-0333-4a1d-a83d-4d8fb90a5315","added_by":"auto","created_at":"2025-06-09 14:56:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":349021,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6796656/v1/bb8bca13da78692bdec88f5a.png"},{"id":84236272,"identity":"f6c62fa7-3738-43b4-9cac-64e117a1149f","added_by":"auto","created_at":"2025-06-09 15:04:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":271599,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6796656/v1/b6e8eec34080d1b02d39db0a.png"},{"id":84236078,"identity":"26f922fc-bbfe-4d99-a728-99668a68358e","added_by":"auto","created_at":"2025-06-09 14:56:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":420411,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6796656/v1/47d69667b1b912a194f407be.png"},{"id":84236274,"identity":"9eca7f8b-e627-41fa-b6f8-6676a57f4c59","added_by":"auto","created_at":"2025-06-09 15:04:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":368336,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6796656/v1/ff18806b735266549fc03bda.png"},{"id":85370507,"identity":"a5232a27-8b58-41e5-9be3-39273bf0e768","added_by":"auto","created_at":"2025-06-25 07:25:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1677304,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6796656/v1/87699c2a-38d1-4323-bca8-725bdd8a6698.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modulation of N-Glycosylation in Bispecific Antibody Biosimilars through Combined Modulators","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe approval and utilization of biosimilars have enhanced the accessibility of medicines and, to some extent, mitigated the rising healthcare costs. Biosimilars exhibit high similarity to their reference biologics and serve as an alternative therapeutic option for patients relying on expensive originator drugs, such as bevacizumab, rituximab, and adalimumab. In 2015, the National Medical Products Administration (NMPA) of China released its inaugural biosimilar guidelines, after which the biosimilar market in China has grown rapidly, with an anticipated market size of \u003cspan\u003e$\u003c/span\u003e5.6\u0026nbsp;billion by 2027[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In accordance with ICH Q6B guidelines and global regulatory requirements, biosimilars must demonstrate a high level of similarity to the reference product with respect to structure, function, and critical quality attributes in order to obtain regulatory approval[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Consequently, conducting a rigorous comparability assessment between biosimilars and their reference products is essential. Due to the proprietary and undisclosed nature of the manufacturing processes for reference drugs, establishing a production process is the initial step in biosimilar development. The production processes of biosimilars inherently differ from those of reference drugs, and variations in manufacturing methods, such as host cells, culture conditions, and purification processes, may lead to differences in the quality attributes of biotechnological products[3\u0026ndash;5]. Biosimilars necessitate comprehensive similarity studies focusing on critical quality attributes, including primary structure (e.g., amino acid sequence and disulfide bonds), higher-order structures, post-translational modifications (such as glycosylation sites and glycans), additional modifications (e.g., oxidation, deamidation, and glycosylation), charge variants, product-related impurities (such as aggregates, fragments, and mismatched disulfide bonds), and biological activity[6\u0026ndash;8].\u003c/p\u003e \u003cp\u003eMost monoclonal antibodies (mAbs) undergo one or more post-translational modifications (PTMs), which are biochemical alterations occurring after protein synthesis. These modifications, either enzymatic or non-enzymatic, affect various amino acid side chains or peptide bonds and include phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and proteolysis[8, 9]. Among these, glycosylation\u0026mdash;defined as the covalent attachment of oligosaccharides to amino acid residues such as asparagine (N-linked) or serine/threonine (O-linked) plays a particularly vital role. It significantly influences a protein's stability, conformation, receptor binding affinity, and biological activity. As such, glycosylation is widely regarded as one of the most critical PTMs in therapeutic protein development[10].Glycosylation is widely present in mammalian cells, for monoclonal antibody (mAb) therapeutics, N-linked glycosylation predominantly occurs at the conserved Asn297 residue in the Fc region[11, 12].\u003c/p\u003e \u003cp\u003eCommon glycan structures found on antibodies include mannose, galactose, fucose, and N-acetylglucosamine. Glycosylation of the Fc (crystallizable fragment) region plays a critical role in modulating interactions with Fc gamma receptors (FcγRs), complement component C1q, and the neonatal Fc receptor (FcRn), thereby influencing antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and serum half-life[11, 13\u0026ndash;15]. Studies have shown that immunoglobulin G (IgG) antibodies lacking Fc glycans lose their capacity to bind FcγRs and C1q, resulting in the complete loss of ADCC and CDC activity. In addition, the absence of glycosylation alters the net charge of the antibody, which can reduce its circulatory half-life [16, 17]. Fucose is typically linked to the innermost N-acetylglucosamine of N-glycans via an α-1,6 linkage. More than 90% of antibodies expressed in Chinese hamster ovary (CHO) cells exhibit core fucosylation. This modification significantly impacts ADCC, as afucosylated IgG antibodies demonstrate up to a 50-fold increase in binding affinity for FcγRIIIa and a 100-fold enhancement in ADCC, without appreciable changes in binding to FcγRI, FcγRIIa, or C1q[18]. In drug development, high-mannose glycoforms are closely monitored due to their considerable influence on pharmacokinetics (PK). Antibodies bearing mannose-rich glycans exhibit faster systemic clearance\u0026mdash;approximately 40% higher\u0026mdash;compared to bisected, fucosylated structures[19]. Galactose is a terminal monosaccharide commonly found on the N-glycan structures of antibodies, and its presence can affect both functional activity and pharmacokinetics. Increased galactosylation has been shown to enhance binding to complement component C1q, thereby promoting CDC. For example, galactosylation of the Fc region of rituximab correlates with improved CDC activity[20, 21]. However, galactose modification appears to have minimal influence on antibody half-life. Comparative studies of antibodies with different levels of galactosylation, such as G0F and G2F glycoforms, have demonstrated no significant differences in their in vivo pharmacokinetics[22, 23].\u003c/p\u003e \u003cp\u003eGlycosylation of monoclonal antibodies is a highly heterogeneous and tightly regulated post-translational modification that is critically influenced by various upstream process parameters. Among the most significant factors are the host cell line, culture medium composition, and cell culture process conditions, all of which can profoundly alter the structure and distribution of N-glycans on the Fc region of antibodies[24, 25]. The choice of expression system, particularly the host cell line, plays a central role in determining glycosylation patterns, CHO cells are widely used in therapeutic antibody production due to their human-compatible glycosylation capabilities[26]. During cell line development, genetic engineering techniques can be employed to modulate the expression of glycosyltransferases, thereby significantly altering the glycosylation patterns of antibodies. For example, knockout of the FUT8 gene in CHO cells effectively reduces core fucosylation, resulting in enhanced antibody-dependent cellular cytotoxicity (ADCC) activity[27]. Additionally, the use of endogenous internal ribosome entry sites (IRES) and polycistronic vectors to co-regulate the expression of α-mannosidase II (MANII) and β-1,4-N-acetylglucosaminyltransferase III (cGNTIII) has enabled the production of antibodies with distinct glycan profiles, leading to improved effector functions[28].Cell culture conditions, including media composition[29], the use of additives[30], pH[31], dissolved oxygen levels[32], culture temperature, and feeding strategies[33, 34], can indirectly influence glycosylation by altering cellular metabolism and the activity of glycosylation enzymes in the endoplasmic reticulum and Golgi apparatus. In recent years, advances in process analytical technologies (PAT)[35] and predictive modeling have also contributed to glycan modulation[36]. Among these approaches, the use of additives epresents one of the simplest and most effective strategies for glycan regulation. Small molecule inhibitors of mannosidases I and II, such as kifunensine and 1-deoxymannojirimycin (DMJ) are also widely used[37, 38]. However, these inhibitors degrade rapidly at room temperaturxe and are costly. Previous studies have investigated the regulation of galactosylation using various glycosylation modulators, such as galactose, uridine, manganese ions, and their mixtures[39, 40]. A growing array of culture medium additives, including metal ions, vitamins, sugars, and nucleosides, are being utilized to regulate N-glycosylation of mAbs without compromising other key quality attributes. The addition of different metal ions (Mn\u0026sup2;⁺, Cu\u0026sup2;⁺) and sugars (galactose, glucosamine) to the culture medium was found to significantly influence antibody glycosylation. Galactose supplementation markedly increased galactosylation levels, while Mn\u0026sup2;⁺ and glucosamine enhanced sialylation, collectively contributing to an overall improvement in the glycan profiles[41\u0026ndash;44].\u003c/p\u003e \u003cp\u003eThis research focuses on optimizing the N-Glycan profile of a biosimilar monoclonal antibody to closely match that of the reference product. By first testing individual regulators at different concentrations to evaluate their effects on cell growth, expression, and N-Glycan profiles, the optimal conditions are identified. Subsequently, combinations of these regulators are applied to further refine the glycosylation pattern, aiming for high similarity with the original product. The innovative aspect of this study lies in the strategic use of multiple regulators to achieve precise glycosylation control, ensuring consistency with the reference mAb.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Line and Reagents\u003c/h2\u003e \u003cp\u003eCHO-K1 cells (Merck), stably expressing a bispecific monoclonal antibody, were used for all experiments. The amplification medium (M1), fed-batch basal medium (M2), and feed media (FM1/FM2, mixed at a 10:1 ratio) were obtained from Cytiva. Manganese chloride (MnCl₂), galactose, and Tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma-Aldrich. Glucose was sourced from Shanghai Shenggong.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSeed Expansion\u003c/h3\u003e\n\u003cp\u003eCells were thawed into 125 mL shake flasks with an initial working volume of 25 mL. Cultures were maintained at 36.5\u0026deg;C, 5% CO₂, and 80% relative humidity with shaking at 125 rpm. Cells were passaged every three days at approximately 0.3 \u0026times; 10⁶ cells/mL until the required seed volume was achieved.\u003c/p\u003e\n\u003ch3\u003eShake Flask Culture\u003c/h3\u003e\n\u003cp\u003eCells were inoculated into 250 mL shake flasks at an initial density of ~\u0026thinsp;0.5 \u0026times; 10⁶ cells/mL and a working volume of 50 mL. Culture conditions were as described above. On day 4, the temperature was shifted from 36.5\u0026deg;C to 32.0\u0026deg;C. The process followed a fed-batch strategy, with FM1 added at 4%, 5%, 5%, 5%, and 4% (v/v) of the initial culture volume on days 3, 6, 8, 10, and 12, respectively; FM2 was supplemented at 10% of the FM1 volume. Stock solutions of 400 mM galactose and 500 \u0026micro;M MnCl₂ were prepared. Galactose was added at final concentrations of 4, 8, and 10 mM, and MnCl₂ at 0.10, 0.25, and 0.40 \u0026micro;M, on each feeding day. Cultures were harvested on day 14. Daily samples were collected for cell density and metabolite analysis. At harvest, cultures were centrifuged at 7000 rpm for 15 minutes, and supernatants were filtered through 0.22 \u0026micro;m filters for subsequent purification, titer determination, and quality assessments.\u003c/p\u003e\n\u003ch3\u003eBioreactor Culture\u003c/h3\u003e\n\u003cp\u003eCells were inoculated into a 2 L bioreactor at an initial density of 0.5 \u0026times; 10⁶ cells/mL, with a starting volume of 1.4 L. The pH was controlled at 6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, with dissolved oxygen maintained at 40%, and agitation set to 180 rpm. The feeding strategy and temperature shift followed the shake flask protocol. 500 mM Tris stock solution was prepared, and final concentrations of 0.5, 0.75 and 1.0 mM Tris were added on feeding days. 1 M galactose stock solution was also prepared, and 4, 8 and 12 mM galactose was added on feeding days. On non-feeding days, glucose concentration was maintained above 6 g/L by supplementing to 8 g/L when necessary. On feeding days, if glucose dropped below 6 g/L, it was replenished to 10 g/L. Daily samples were taken for cell growth, metabolic profiling, and osmolality measurements. The culture was harvested on day 14, followed by centrifugation at 7000 rpm for 15 minutes. Supernatants were filtered through 0.22 \u0026micro;m filters and used for downstream purification and quality analysis.\u003c/p\u003e\n\u003ch3\u003eProtein Purification, Titer, and Quality Analysis\u003c/h3\u003e\n\u003cp\u003eClarified supernatants were purified by Protein A affinity chromatography using AT ProteinA Diamond Plus resin. The purified antibodies were subjected to quality assessments. Titer was quantified via Protein A affinity high-performance liquid chromatography (HPLC). Purity was evaluated using size-exclusion HPLC (SE-HPLC) with a TOSOH TSKgel G3000 SWXL column and a neutral pH mobile phase, with UV detection. Charge variants were assessed using cation-exchange HPLC (CE-HPLC) on a Thermo ProPac WCX-10 (4 \u0026times; 250 mm) column, also with UV detection. For N-glycan profiling, glycans were enzymatically released using PNGase F, derivatized with 2-aminobenzamide (2-AB), and analyzed by hydrophilic interaction liquid chromatography (HILIC).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe biosimilar Bs-mAb1 showed significant differences in glycosylation patterns\u0026mdash;particularly in mannose, galactose, and fucose compared to the reference product. Specifically, mannose and galactose glycosylation levels were reduced by 113.7% and 90%, respectively, while fucose glycosylation was 5.0% higher. To align the glycosylation profile of the biosimilar more closely with that of the reference product, this study evaluated the effects of galactose and MnCl₂, aimed at enhancing galactosylation, and Tris, intended to modulate mannose and fucose glycosylation. Their impacts on cell growth, metabolism, antibody yield, and overall product quality were systematically assessed.\u003c/p\u003e \u003cp\u003eBased on previous reports and internal platform data, galactose was evaluated at concentrations of 4 mM, 8 mM, and 12 mM, and MnCl₂ at 0.10 \u0026micro;M, 0.25 \u0026micro;M, and 0.40 \u0026micro;M. The impact on cell growth, metabolism, antibody titer, and particularly N-glycan profiles was assessed. Viable cell density and viability (Fig.\u0026nbsp;1) showed similar peak densities across groups, with cell viability maintained above 95% at harvest. Lactate metabolism (Fig.\u0026nbsp;1b) revealed that lactate levels steadily declined across all galactose concentrations. Titer data (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) showed that the control group reached 6.1 g/L, while the galactose-treated groups maintained titers around 6.0 g/L, indicating that galactose supplementation had no adverse effects on growth, metabolism, or antibody production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProduct quality analysis (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed no significant differences in main peak, acidic variants, or mispaired peaks between the galactose-treated and control groups. SE-HPLC (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) confirmed consistent main peak content, suggesting no impact on purity. N-glycan analysis (Fig.\u0026nbsp;1) demonstrated that galactose supplementation significantly increased galactosylation without affecting mannose or fucose levels. Galactose concentrations of 4 mM, 8 mM, and 12 mM led to increases of 42.3%, 83.8%, and 85.9%, respectively, in galactosylation, exhibiting a dose-dependent effect that plateaued between 8 mM and 12 mM\u0026mdash;consistent with previous studies. Based on these findings and the gap in galactosylation between the biosimilar and reference product, 8 mM galactose was selected for subsequent bioreactor studies.\u003c/p\u003e \u003cp\u003eIn contrast, MnCl₂ at higher concentrations negatively impacted cell proliferation. As shown in Fig.\u0026nbsp;2, the VCDEnd in the control group reached 26.0 \u0026times; 10⁶ cells/mL, whereas the 0.40 \u0026micro;M MnCl₂ group achieved only 19.0 \u0026times; 10⁶ cells/mL. Elevated MnCl₂ also led to increased lactate accumulation in the late culture phase (Fig.\u0026nbsp;2), indicating metabolic stress. Titer data (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) showed that antibody yield in the high MnCl₂ group was approximately 30% lower than in the control. However, CE-HPLC (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and SE-HPLC (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) analyses indicated that MnCl₂ had no significant effect on charge variants or aggregation.\u003c/p\u003e \u003cp\u003eN-glycan analysis (Fig.\u0026nbsp;1f) revealed that MnCl₂ had a pronounced effect on galactosylation, with increases of 241.7%, 399.0%, and 612.7% observed at 0.10 \u0026micro;M, 0.25 \u0026micro;M, and 0.40 \u0026micro;M, respectively. This enhancement was dose-dependent, while mannose and fucose levels remained unchanged. Due to the excessive galactosylation and the detrimental impact on cell growth and antibody expression, MnCl₂ was not pursued in further bioreactor studies.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Effects of Galactose and MnCl\u003csub\u003e2\u003c/sub\u003e on Titer and purity\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\"\u003e\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\u003cstrong\u003eTable 2\u0026nbsp;\u003c/strong\u003eEffects of Galactose and MnCl\u003csub\u003e2\u003c/sub\u003e on antibody heterosomes\u003c/div\u003e\n\u003cdiv\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"533\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eItem\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eJJ\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4484%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eacidic\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e\u003cstrong\u003emain\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e\u003cstrong\u003ebasic\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.1332%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eQQ\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003e%\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e22.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4484%;\"\u003e\n \u003cp\u003e2.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e63.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e9.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.1332%;\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e4mM Galactose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e20.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4484%;\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e64.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.1332%;\"\u003e\n \u003cp\u003e1.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e8mM Galactose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e22.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4484%;\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e62.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e11.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.1332%;\"\u003e\n \u003cp\u003e1.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e12mM Galactose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e23.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4484%;\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e60.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e10.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.1332%;\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e0.10\u0026mu;M MnCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e21.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4484%;\"\u003e\n \u003cp\u003e2.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e63.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e11.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.1332%;\"\u003e\n \u003cp\u003e1.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e0.25\u0026mu;M MnCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e25.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4484%;\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e59.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.1332%;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e0.40\u0026mu;M MnCl\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.9493%;\"\u003e\n \u003cp\u003e22.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 17.4484%;\"\u003e\n \u003cp\u003e2.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e62.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 15.7598%;\"\u003e\n \u003cp\u003e10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 13.1332%;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003eShake flask models have limitations due to the absence of pH and dissolved oxygen (DO) control, which can affect viable cell density (VCD) and cell viability. Additionally, studies have shown that Tris can exert cytotoxic effects at certain concentrations, especially under suboptimal pH conditions. Therefore, to more accurately evaluate the effects of Tris, experiments were conducted in a 2 L stirred-tank bioreactor, testing concentrations of 0.50 mM, 0.75 mM, and 1.00 mM. The impact on antibody glycosylation, cell growth, and metabolism was assessed.\n\u003c/div\u003e\n\u003cp\u003eAs shown in Fig. 3, different concentrations of Tris had no significant effect on cell growth, and lactate metabolism patterns remained consistent across groups. HPLC analysis (Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) indicated that the main peak content of the antibody remained around 95% across all conditions, suggesting no impact on product purity. Furthermore, CE-HPLC data (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) confirmed that Tris did not alter the formation of acidic variants compared to the control.\u003c/p\u003e\n\u003cp\u003eN-glycan analysis (Fig. 3) revealed a positive correlation between Tris concentration and mannose glycosylation, and a negative correlation with fucose glycosylation, while the impact on galactosylation was minimal. Specifically, at 0.50 mM, 0.75 mM, and 1.00 mM Tris, fucose glycosylation decreased by 2.9%, 5.6%, and 10.2%, respectively, while mannose glycosylation increased by 61.9%, 114.3%, and 204.8%. Tris primarily reduced G0F structures and increased the proportion of Man5. Among the tested concentrations, 0.75 mM Tris yielded a glycosylation profile most similar to the reference product without compromising cell growth, metabolism, or other quality attributes. Therefore, 0.75 mM Tris was selected for further study.\u003c/p\u003e\n\u003cp\u003eWhen 0.75 mM Tris and 8 mM Galactose were added in combination, cell growth and metabolism remained unaffected (Fig. 4), except for a slight reduction in glucose consumption compared to the control. This was likely due to galactose serving as an alternative energy source, reducing glucose demand. CE-HPLC results (Table 4) confirmed that the combination of Tris and Galactose did not impact charge variant formation, and SE-HPLC data (Table 3) showed no effect on antibody purity.\u003c/p\u003e\n\u003cp\u003eN-glycan analysis (Fig. 4) demonstrated that combined supplementation with Tris and Galactose resulted in a 118.2% increase in mannose glycosylation, a 78.8% increase in galactose glycosylation, and a 6.2% decrease in fucose glycosylation. Overall, this combination produced a glycosylation profile highly consistent with that of the reference product.\u0026nbsp;\u003c/p\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of Tris on Titer and purity\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eItem\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTiter(g/L)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLW(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eHW(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMain(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN.D.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5mM Tris\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN.D.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75mM Tris\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN.D.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1mM Tris\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN.D.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75mM Tris\u0026thinsp;+\u0026thinsp;8mM Galactose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eN.D.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e96.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffects of Tris on antibody heterosomes\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eItem\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eJJ(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eacidic(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003emain(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ebasic(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eQQ(%)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e57.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5mM Tris\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75mM Tris\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0mM Tris\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75m MTris\u0026thinsp;+\u0026thinsp;8mM Galactose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n"},{"header":"Discussion","content":"\u003cp\u003eGlycosylation is a critical quality attribute (CQA) of monoclonal antibodies, affecting their efficacy, stability, and immunogenicity. In this study, we systematically assessed the effects of galactose, MnCl₂, and Tris on the N-glycan profile, cell growth, metabolism, and product quality of a bispecific monoclonal antibody biosimilar. These additives were selected based on their established or proposed roles in modulating specific glycosylation pathways, with the goal of improving similarity to the reference product.\u003c/p\u003e \u003cp\u003eGalactose supplementation enhanced galactosylation in a clear dose-dependent manner, with little to no impact on other glycoforms or cell culture performance. This is consistent with previous findings that exogenous galactose serves as a substrate for β-1,4-galactosyltransferase in the Golgi, increasing terminal galactose residues on N-glycans[26]. A saturation point was observed between 8 mM and 12 mM, suggesting limitations in enzyme capacity or intracellular transport[45]. Notably, galactose addition did not negatively affect cell viability, lactate metabolism, titer, or other quality attributes, making it a straightforward and effective strategy for enhancing galactosylation in biosimilar development.MnCl₂ had a more complex impact. While it significantly increased galactosylation in a dose-dependent fashion, it also impaired cell growth and productivity at higher concentrations. This is likely due to manganese\u0026rsquo;s dual role as a cofactor for glycosyltransferases, and as a cytotoxic agent when present at elevated levels[46, 47]. The accumulation of lactate and reduced viable cell density in MnCl₂ treated cultures suggest that oxidative or metabolic stress may contribute to its cytotoxicity. Given these drawbacks, MnCl₂ is less ideal for process development unless its concentration is carefully optimized.\u003c/p\u003e \u003cp\u003eTris, a common buffering agent, showed distinctive regulatory effects on mannose and fucose glycosylation. Increasing Tris concentrations were associated with higher mannose levels and reduced fucosylation. This may be due to Tris altering intracellular pH or influencing the activity of glycosylation enzymes such as α-mannosidase II and fucosyltransferase 8 (FUT8)[48, 49]. Although Tris can be cytotoxic under certain conditions[50], no adverse effects on cell growth, viability, or antibody quality were observed in the bioreactor system, likely due to well-controlled pH and dissolved oxygen levels.The combination of 0.75 mM Tris and 8 mM galactose produced a synergistic effect, significantly improving the glycosylation profile in terms of galactose, mannose, and fucose content, without negatively impacting cell growth, metabolism, or product quality. The absence of interference between the two additives further supports the viability of combined additive strategies in upstream process optimization. Overall, these findings provide practical insights into the targeted modulation of N-glycosylation through small-molecule additives. This strategy represents a flexible, patent-friendly, and cost-effective alternative to genetic engineering, particularly valuable in biosimilar development where close glycan similarity to the reference product is required. In addition, the experimental approach, which involves a systematic evaluation of both individual and combined additives under well-controlled bioreactor conditions, serves as a valuable reference for future glycoengineering efforts during upstream process development.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eGlycosylation modifications of monoclonal antibodies and recombinant proteins are critical quality attributes that have a significant impact on drug efficacy, safety, and half-life. Key factors such as the glycosylation site, glycan type, and glycan abundance influence the biological activity, stability, and overall performance of the product. During upstream process development, glycosylation can be modulated through careful cell line selection, genetic engineering, process parameter optimization, media formulation, and feeding strategies. Although genetic modification provides precise control, process optimization and the use of regulatory additives are often preferred in industrial settings due to patent constraints and practical considerations. Among these strategies, the addition of specific small molecules represents a cost-effective and efficient approach to modulate glycosylation, particularly in biosimilar development, where close similarity to the reference product is essential.In this study, we systematically evaluated the effects of galactose, MnCl₂, Tris, and their combinations on cell growth, metabolism, antibody yield, and N-glycan profiles. Galactose and Tris, whether used individually or together, showed no adverse effects on cell performance, whereas MnCl₂ reduced both cell growth and protein expression. Notably, the combination of 8 mM galactose and 0.75 mM Tris significantly improved the glycosylation pattern. Mannose glycosylation increased by 118.2%, galactose by 78.8%, and fucose levels decreased by 6.2%, with no evidence of negative interaction between the two additives. The resulting N-glycan profile closely resembled that of the reference product.This work demonstrates a practical and effective approach for controlling glycosylation using small-molecule additives, offering a valuable reference for accelerating biosimilar development and reducing production costs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no financial or non-financial interests that could be perceived as directly or indirectly influencing the work submitted for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWei, K.K., et al., Biosimilars: navigating the regulatory maze across two worlds. 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TOXICOLOGY AND INDUSTRIAL HEALTH, 2024. 40(7): p. 387-397.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Glycosylation, Biosimilars, Monoclonal Antibodies, Additives, Cell culture","lastPublishedDoi":"10.21203/rs.3.rs-6796656/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6796656/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo ensure a high degree of similarity between biosimilars and reference drugs, it is crucial to perform comprehensive characterization and maintain rigorous control over glycosylation processes. Here,we aimed to optimize the glycosylation profile of a biosimilar of a bispecific monoclonal antibody (Bs-mAb1) to closely resemble that of the reference drug through the synergistic use of glycosylation modulators. To identify the strongest modulators and appropriate concentration ranges, we first examined the effects of different concentrations of galactose (Gal) and manganese chloride (MnCl\u003csub\u003e2\u003c/sub\u003e) on the galactosylation rate in Shake Flask, as well as the influence of tris(hydroxymethyl)aminomethane (Tris) on the incorporation of mannose and fucose in 2 L Bioreactor. Importantly, the concurrentuse of Tris and galactose did not result in any interaction effects on N-glycan modifications and had no detrimental impact on cell growth, metabolism, antibody charge variants or purity. In conclusion, The concurrent use of 0.75 mM Tris and 8 mM galactose yields a glycosylation profile of Bs-mAb1 that is highly comparable to that of the reference drug, thereby providing an effective strategy for optimizing glycosylation in biosimilars. These findings provide significant insights into the regulation of glycosylation in the production of therapeutic monoclonal antibodies and may contribute to enhancing the consistency and therapeutic performance of biosimilars.\u003c/p\u003e","manuscriptTitle":"Modulation of N-Glycosylation in Bispecific Antibody Biosimilars through Combined Modulators","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-09 14:56:54","doi":"10.21203/rs.3.rs-6796656/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a91cba1b-3304-4e3c-a517-3cc1f9f7fdd4","owner":[],"postedDate":"June 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-25T07:24:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-09 14:56:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6796656","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6796656","identity":"rs-6796656","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}