(52d) Differences in Recombinant Monoclonal Antibody Glycosylation Between Stable and Transient Gene Expression Systems Conference: AIChE Annual MeetingYear: 2014Proceeding: 2014 AIChE Annual MeetingGroup: Food, Pharmaceutical & Bioengineering DivisionSession: Cell Culture I: Process and Media Design Time: Monday, November 17, 2014 - 9:24am-9:42am Authors: Sou, S. N., Imperial College London Sellick, C., MedImmune Lee, K., MedImmune Mason, A., MedImmune Polizzi, K. M., Imperial College London Kontoravdi, C., Imperial College London Transient gene expression (TGE) can rapidly deliver recombinant proteins within 10 – 18 days as opposed to traditional stable gene expression (SGE) systems which require several weeks of cell line development. TGE is especially useful for quick provision of materials for pre-clinical trials, research and some early-stage clinical evaluations. However, the low recombinant protein titre is a significant limitation of this method. As a result, there is increased focus on optimising TGE production yields to attain stable system levels. However, few studies have directly compared the glycan contents of mAbs between stable and transient gene expression. mAb glycosylation, which has widely been reported to affect drug safety and efficacy, has to be comparable between the two expression systems. This work compared the quality of SGE- and TGE-derived mAbs produced under mild hypothermic conditions by studying their respective cell metabolic behaviour. The specific productivity of mAb (qmab) generated from the TGE under mild hypothermic conditions was around 28% lower than that from the stable transfectants at the same temperature, accompanied by reduced expression of heavy and light chains at both the mRNA and the peptide level. Unlike the SGE where there was rapid CHO cell growth, viable cell density was maintained at a constant level in the TGE. Increased in cell metabolism was observed where glucose consumption per cell was higher in the TGE than in SGE. With an abundance of intracellular glucose, the synthesis of nucleotide sugar donors was increased in the TGE system. Intracellular concentrations of UDP-Glc, UDP-GlcNAc and UDP-Gal were higher in cells under TGE than in SGE at 32°C. mAb glycosylation also varied between the two systems. The TGE system produced an increased proportions of both less processed (Man5, G0) and more processed (G2, G2F) glycan structures, but reduction in the intermediate glycoforms (G0F). Results suggested that mAb galactosulation was more readily achieved in the TGE system. On the other hand, results from flux balance analysis showed that at late exponential phase, transiently transfected CHO-cells consumed higher amount of glucose, where more energy was used for nucleotide, NSD and lipid syntheses when compared to stable transfectants. Our study demonstrates the impact of transient gene expression on product glycosylation. Based on our understanding of the difference in CHO cell metabolic flux distribution between TGE and SGE, we aim to recreate glycan pattern of stably expressed therapeutic protein in the TGE system through media optimization.