Glycosylation, a pivotal post-translational modification, serves as an essential critical quality attribute (CQA) for therapeutic antibodies, profoundly regulating their biological function, structural stability, protein folding, immunogenicity, and effector activities. While numerous studies have explored the effects of process parameters (PPs) on glycosylation variability, the absence of standardized reporting protocols has hindered cross-study comparisons and precluded definitive, universal conclusions. The standardized Glycan Index (GIx) enables systematic reintegration and quantitative meta-analysis of published data, allowing objective evaluation of how bioprocess parameters modulate glycan distribution profiles in Chinese hamster ovary (CHO) cell-derived monoclonal antibodies (mAbs). This review systematically elaborates the regulatory effects of culture temperature, pH, dissolved oxygen, partial pressure of carbon dioxide, osmolality, and chemical additives on antibody glycosylation.
1. Regulatory Effects of Temperature Modulation on Glycosylation
Temperature modulation during the production phase is a prevalent and well-established process strategy widely applied in industrial biopharmaceutical manufacturing.
Typical temperature control strategies fall into three categories: (1) constant low-temperature cultivation from the initial culture stage; (2) temperature downshift from 37 °C to 30–35 °C in the late cultivation phase; (3) periodic temperature cycling cultivation.
Accumulated studies have verified that reduced culture temperature alters cellular galactosylation and fucosylation levels, thereby modulating the biological activity of therapeutic antibodies. A statistical analysis of nine relevant studies showed that GIx increased by ≥10% in one study and decreased by ≥10% in three studies, among which only one reported a pronounced reduction. Most remaining cases exhibited marginal changes in galactosylation, while sialylation levels remained largely stable.
The temperature-dependent modulation of glycosylation can be mechanistically explained by multiple cellular adaptive responses to hypothermia. Low-temperature culture suppresses cellular metabolism and proliferation, remodels the expression profile of nucleotide-sugar transporters and glycosyltransferases, and reduces the secretion of endogenous non-target proteins. This mitigates intracellular resource competition between endogenous proteins and recombinant mAbs, indirectly affecting glycan modification efficiency.
Additionally, reduced culture temperature commonly elevates specific productivity (qP) of mAbs. The enhanced specific production rate shortens the residence time of antibodies in the secretory pathway. Under conditions of limited glycan precursor supply, this truncated trafficking process reshapes the final glycan signature of secreted antibodies.
2. Regulatory Effects of pH Control on Glycosylation
pH is a core process parameter for cell culture, routinely maintained at approximately 7.0 via carbon dioxide aeration or alkali supplementation, with sodium hydroxide, sodium carbonate, and sodium bicarbonate serving as common pH regulators. Notably, pH deviation from the baseline value does not trigger consistent, predictable glycosylation responses.
Elevated pH (>7.0) has been reported to either increase or decrease glycan heterogeneity in different culture systems, leading to contradictory phenotypic outcomes. When culture pH drops below 7.0, the galactosylation index (GI) increases by 2.32% while the sialylation index (SI) decreases by 0.7%; in contrast, alkaline pH (>7.0) exerts negligible impacts on these two key glycosylation indices.
Conflicting pH-induced glycosylation alterations have been observed even with identical cell lines and mAb products, making it challenging to establish a universal regulatory framework. Where pH-induced GIx variations occur, changes are predominantly confined to galactosylation, with the overall ΔGIx generally lower than 10%.
Mechanistically, mildly acidic extracellular conditions (e.g., pH 6.7) induce cytoplasmic alkalinization and disrupt the physiological pH gradient across the endoplasmic reticulum (ER) and Golgi apparatus lumen. Meanwhile, acidic pH accelerates vesicular trafficking from the ER to the Golgi complex, shortening the retention time of nascent proteins in the secretory pathway. This results in the premature secretion of proteins with incompletely processed glycans, ultimately altering cellular glycosylation patterns.
3. Regulatory Effects of Dissolved Oxygen and Carbon Dioxide Tension on Glycosylation
Dissolved oxygen (DO), monitored as dissolved oxygen tension (DOT), is a vital process parameter typically controlled at 20%–50% air saturation in CHO cell culture. Oxygen transfer efficiency is determined by multiple operational factors, including gas flow rate, inlet oxygen partial pressure, bubble size, agitation speed, and impeller configuration. Hypoxia induced by insufficient DO and oxidative stress triggered by excessive DO both elevate reactive oxygen species (ROS) levels, impairing cell growth, viability, mAb productivity, and product quality.
To date, only a limited number of systematic studies have investigated DO-mediated glycosylation regulation. Most published data demonstrate minimal GIx fluctuations with ΔGIx < 1.0%. Antioxidant supplementation (e.g., baicalein, S-sulfocysteine) is commonly adopted to alleviate high DO-induced oxidative stress and ROS accumulation. However, media fortified with these antioxidants showed no significant changes in glycosylation indices when cells were cultured under 50% air saturation.
Partial pressure of carbon dioxide (pCO₂) modulates cell culture performance primarily through its regulatory effect on pH homeostasis. Mammalian cell cultures are conventionally maintained at 5% CO₂, with fine-tuning applied to stabilize culture pH. The molecular mechanism underlying pCO₂-regulated mAb glycosylation remains poorly elucidated, largely due to the indirect crosstalk between pCO₂ and other process parameters. Existing studies indicate that pCO₂ elevation above the conventional 5% level slightly reduces GI by less than 5%, with no observable impacts on fucosylation or sialylation levels.
4. Regulatory Effects of Osmolality on Glycosylation
Osmolality is a critical process parameter that modulates cell growth, viability, and specific productivity. Culture osmolality gradually increases during fed-batch cultivation due to continuous feed supplementation and accumulation of metabolic byproducts, with the optimal range for mammalian cell culture maintained at 280–320 mOsm/kg. Hyperosmotic stress induced by NaCl supplementation effectively enhances mAb specific productivity, while significantly altering glycosylation profiles.
Published data confirm that elevated osmolality reduces GI by approximately 10% and decreases the fucosylation index (FI) by 2%–10%. These pronounced alterations in galactosylation and fucosylation substantially affect the in vivo therapeutic efficacy of mAbs.
From a mechanistic perspective, hyperosmotic stress downregulates the expression of key genes involved in glycosylation, including those encoding nucleotide-sugar transporters and core glycosyltransferases. This transcriptional suppression reduces the intracellular availability of glycosylation substrates and cofactors, thereby attenuating glycan processing and maturation. This mechanism explains the disrupted glycosylation patterns caused by osmolality fluctuations in intensified fed-batch cultures. Accordingly, when improving mAb productivity via hyperosmotic culture, targeted process control strategies are required to guarantee consistent glycosylation quality and batch-to-batch stability.
5. Regulatory Effects of Chemical Additives on Glycosylation
A variety of chemical additives are supplemented in mammalian cell culture systems to enhance cell productivity and mAb yield, including cyclin-dependent kinase (CDK) inhibitors for cell cycle arrest, chemical chaperones, ER stress inhibitors, and histone deacetylase (HDAC) inhibitors such as sodium butyrate and valerate.
Chemical additives exhibit variable modulatory effects on galactosylation. Approximately half of the reported cases show a 6% GIx variation, while the remaining cases present negligible changes with ΔGIx < 1.5%. Notably, these additives generally exert no significant influence on fucosylation levels. Sodium butyrate remodels the transcription of multiple glycosylation-related genes, including the manganese transporter gene slc39a8, yet such transcriptional alterations are insufficient to induce substantial changes in mAb glycosylation signatures.
CDK4/6 inhibitors induce G0/G1 cell cycle arrest in CHO cells, increasing galactose and fucose occupancy on mAb glycans with a ΔGIx of 2%–5%. The combined application of dimethyl sulfoxide (DMSO) and BiP inducer X (BIX) exerts dual effects: it acts as a chemical chaperone and relieves DMSO-induced ER stress, thereby upregulatingB4galt expression and improving glycan modification efficiency. Forskolin supplementation elevates mAb qP by triggering G0/G1 arrest and inhibitingBcl-2 expression. Although forskolin enhances terminal galactosylation, GIx quantification confirms that this modulatory effect is marginal.
Overall, the efficacy of chemical additives is highly process-dependent, and the molecular linkage between additive intervention and glycan remodeling remains to be further clarified.
6. Summary and Conclusion
Conventional bioprocess parameters can modulate CHO cell glycosylation, yet most environmental perturbations induce minor, unstable, and poorly reproducible glycan variations, limiting their application as primary glycoengineering strategies. Standardized quantitative analytical tools represented by GIx provide a robust platform for objective cross-study comparison of process parameter effects on mAb glycosylation. In most scenarios, process fluctuations alter FI, GI, and SI by less than 10%, with the majority of variations below 5%. Nevertheless, extreme deviations in temperature and osmolality can trigger biologically meaningful changes in fucosylation and galactosylation, which profoundly affect mAb functional properties.
Notably, process-induced glycosylation alterations are highly heterogeneous across cell lines, mAb products, and culture modes, resulting in inconsistent glycan profiles even under identical process settings. Therefore, sole optimization of conventional process parameters cannot reliably achieve targeted, stable glycosylation profiles. A more rational and effective strategy involves stable process control within a defined operating region or quality-by-design (QbD) design space, combined with targeted media optimization (e.g., supplementation of galactose, Mn²⁺, uridine, trace elements, and hormones). Furthermore, host cell genetic engineering represents a more precise and predictable approach for glycosylation regulation, including FUT8 knockout, glycosyltransferase modification, and nucleotide-sugar transporter engineering.
