Introduction: The biopharmaceutical industry has witnessed substantial advancements in the production of therapeutic antibodies, particularly those targeting immune-related diseases such as autoimmunity and cancers. Key to the efficacy and specificity of these monoclonal antibodies is their glycosylation profile, which is influenced by various biosynthetic mechanisms. This study specifically examines the impact of culture duration and manganese chloride (Mn2+) concentration on the glycan profile of rituximab, a widely used therapeutic antibody produced in Chinese Hamster Ovary (CHO) cells.
Methods: Both upstream and downstream processes were methodically designed to assess the glycosylation changes in relation to these process parameters. CHO (DG44) cells were cultured in a defined protein-free medium (MAMPF77) under controlled conditions. The cultures were categorized based on varying Mn2+ concentrations (15 mM and 35 mM) and cultivation durations (300 hours vs. 264 hours). Our results indicated that optimized culture conditions can significantly enhance galactosylation of rituximab, achieving increases of up to 8% in the glycan profile.
Harvesting was performed through depth filtration, followed by chromatographic purification techniques, including affinity chromatography, which retained high selectivity and resolution. Subsequent viral inactivation and a series of analytical tests confirmed the integrity and purity of the final product, with impurity levels remaining within acceptable ranges as determined by size exclusion chromatography (SE-HPLC) and SDS-PAGE analyses.
Results: The findings highlight a complex interaction between cultivation duration and manganese chloride concentration. While extended cultivation periods increased protein yields, they inversely impacted the extent of galactosylation; reducing the cultivation duration enabled optimal enzyme activity for the maturation of glycosylation pathways. Moreover, the study demonstrates that Mn2+ concentration plays a critical role as a cofactor in enhancing galactosyltransferase activity, thereby promoting higher levels of galactosylated antibody production.
Conclusion: In conclusion, optimizing upstream parameters such as culture conditions effectively influences the therapeutic glycoprotein's glycan profile, ultimately enhancing its clinical efficacy in immunotherapy. This research provides valuable insights into the biotechnological production strategies that can be employed to maximize the therapeutic potential of monoclonal antibodies, setting a foundation for future developments in antibody engineering and production.