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In this study, omics-based analysis tools were used to explore the effect of glucose starvation and culture duration on monoclonal antibody (mAb) production in fed-batch CHO cell culture to gain better insight into how these parameters can be controlled to ensure optimal mAb productivity and quality. Titer and N-glycosylation of mAbs, as well as proteomic signature and metabolic status of the production cells in the culture were assessed. We found that the impact of glucose starvation on the titer and N-glycosylation of mAbs was dependent on the degree of starvation during early stationary phase of the fed-batch culture. Higher degree of glucose starvation reduced intracellular concentrations of UDP-GlcNAc and UDP-GalNAc, but increased the levels of UDP-Glc and UDP-Gal. Increased GlcNAc and Gal occupancy correlated well with increased degree of glucose starvation, which can be attributed to the interplay between the dilution effect associated with change in specific productivity of mAbs and the changed nucleotide sugar metabolism. Herein, we also show and discuss that increased cell culture duration negatively affect the maturation of glycans. In addition, comparative proteomics analysis of cells was conducted to observe differences in protein abundance between early growth and early stationary phases. Generally higher expression of proteins involved in regulating cellular metabolism, extracellular matrix, apoptosis, protein secretion and glycosylation was found in early stationary phase. These analyses offered a systematic view of the intrinsic properties of these cells and allowed us to explore the root causes correlating culture duration with variations in the productivity and glycosylation quality of monoclonal antibodies produced with CHO cells.

Takket være nylige fremskridt inden for den "omic" revolution af Chinese hamster ovary (CHO) celler, er udvikling og produktion af rekombinante terapeutiske proteiner i CHO cellefabrikker begyndt at blive præget med afsæt i systembiologi. En holistisk forståelse af titer og N-glykosylering af et produceret rekombinant protein i forhold til den tilknyttede celle kultiverings proces, samt en genomisk, proteomisk, metabolisk og fysiologisk forståelse, vise sig at være yderst effektiv når der samtidig er fokus på sikkerhed, effektivitet og pris, af det enkelte protein. Ved at kombinere og sammensætte viden indenfor CHO celle kultiverings teknologi, upstream proces udvikling, metabolisk optimering, og glyco-biologi, til et systematisk framework med fokus på kvalitet og kvantitet, kan produktionen af rekombinante terapeutiske proteiner blive optimeret.I det præsenterede arbejde er det seneste know-how indenfor analyse, kontrol og optimering af N-glykosylering, samt indflydelsen af N-glykosylering på terapeutiske proteiner, blevet grundigt revideret og analyseret. Mere specifikt er det blevet undersøgt hvordan man kan kontrollere og optimere N-glykosylering i CHO celler. Hoved fokus i dette PhD projekt har været at udvikle effektive metoder til modelering af N-glycosylering af CHO-producerede recombinante monoklonale antistoffer (mAb), således at ønskede glycosylerings mønstre kan opnåes, samtidig med at udvide forståelsen for de dybere mekanismer der styrer N-glycosylering set fra et systembiologisk perspektiv. To forskellige strategier blev brugt til at glyko-optimere med stor succes: 1) optimering af medie og kultiverings processer; 2) genetisk forbedring af CHO som cellefabrik.I den første del af tesen, er den første strategi demonstreret af flere successfulde case-studies, hvor process- og medieoptimering blev brugt til at styre N-glykosyleringen. Balancen mellem glukose- og aminosyremetabolismen blev kontrolleret ved at bruge galaktose som feed additiv og ved at ændre process parametre såsom udsåningstætheden og længden af kultiveringen. Ved at kontrollere balancen mellem glukose- og aminosyremetabolismen, kunne N-glycosyleringen påvirkes. Der er flere forklaringer på, hvorfor denne balance styrer glykosyleringen, herunder mekanismer associeret med produktion, metabolisme, proteomet, og fysiologi.I den anden del af tesen bliver både literaturen og eksperimentelle applikationer undersøgt, for at demonstrere hvorledes omics data og implementering af systembiologi kan udnyttes til at forstår biologiske mekanismer, herunder N-glycosylering i CHO celler.I den tredje og sidste del af tesen, bliver den anden strategi demonstreret. Lovende resultater viser at det er muligt at optimere N-glykosylering ved at modificere genetikken og metabolismen i CHO celler. Ved at overudtrykke enten N-acetylglucosaminyltransferase I (GnTI) proteiner kunne GlcNAc tilgængeligheden øges, med en medfølgende forbedring af matureringen af glykaner i mAbs.Resultaterne demonstrerer integrationen af systembiologi koncepter og process- og metabolisme modifikationer, som en effektiv måde hvorved N-glykosylering af CHO-producerede rekombinante terapeutiske proteiner kan kontrolleres og optimeres. Thanks to the recent advances in Chinese hamster ovary (CHO) “omic” revolution, the development of recombinant therapeutic protein bioprocessing using CHO cell factory started to merge with the new biological mindset called systems biology. In order to produce a CHO-derived recombinant therapeutic protein with ensured safety, efficacy and cost-effectiveness, holistic understanding of titer and N-glycosylation of the protein in relation to cell culture process as well as genomic, proteomic, metabolic and physiological status of the cells becomes a superior approach. Combining the knowledge of CHO cell culture technology, upstream process engineering, metabolic engineering, and glycobiology into a systematic framework allow us to improve the production of recombinant therapeutic protein towards an optimal balance between quantity and quality.In the presented work, recent know-how on impact, analysis, control and optimization of N-glycosylation were thoroughly reviewed. In particular, how to control and optimize N-glycosylation in CHO cells was exclusively studied. The main focus of this PhD project is to find effective approaches of modulating N-glycosylation of CHO-derived recombinant monoclonal antibody (mAb) towards desired patterns, and at the same time try to understand the underlying mechanisms of that from a systems biology perspective. Two different strategies were used and achieved great success in glyco-optimization: 1) optimize media and culture process; 2) Genetically optimize CHO cell factory.In the early part of the thesis, the first strategy was displayed by a number of successful case studies, in which process and media engineering approach was successfully used to direct N-glycosylation. Controlling the balance between glucose and amino acid metabolism, using galactose as feed additives, changing process parameters such as seeding density and cultivation duration are all demonstrated to be effective. The causal explanation of their impact on glycosylation can be various, including product, metabolism, proteome and physiology-associated mechanism.In the middle part of the thesis, both literature reviews and experimental applications were provided to demonstrate how to use omics data and implement systems biology to understand biological activities, especially N-glycosylation in CHO cells.In the last part of the thesis, the second strategy that apply genetic and metabolic engineering approach to improve N-glycosylation capability of CHO cells was also presented promising results. Overexpression of either N-acetylglucosaminyltransferase I (GnTI) in CHO cells was confirmed to improve the maturation of glycans in mAb.In conclusion, integrating the concept of systems biology with process and metabolic engineering has been demonstrated through a number of studies to be a superior way of controlling and optimizing N-glycosylation of CHO-derived recombinant therapeutic protein.