000135629 001__ 135629
000135629 005__ 20180501105929.0
000135629 0247_ $$2doi$$a10.5075/epfl-thesis-4393
000135629 02470 $$2urn$$aurn:nbn:ch:bel-epfl-thesis4393-7
000135629 02471 $$2nebis$$a5759988
000135629 037__ $$aTHESIS_LIB
000135629 041__ $$aeng
000135629 088__ $$a4393
000135629 245__ $$aTransient recombinant protein expression in mammalian cells$$bthe role of mRNA level and stability
000135629 269__ $$a2009
000135629 260__ $$aLausanne$$bEPFL$$c2009
000135629 300__ $$a145
000135629 336__ $$aTheses
000135629 520__ $$aTransient gene expression (TGE) is a rapid method for generating recombinant proteins in mammalian cells, but the volumetric productivities for secreted proteins in transiently transfected CHO DG44 cells are typically more than an order of magnitude lower than the yields achieved with recombinant CHO-derived cell lines. The goals of the thesis are to identify the limitations to higher TGE yields in CHO DG44 cells and to find possible solutions to overcome the problems. Initially an attempt was made to enhance TGE production by increasing the amount of transfected plasmid DNA. However, this approach did not result in increased recombinant protein levels; on the contrary, transfection with an excess of plasmid DNA (> 1.25 μg/ml) had a negative impact on transgene mRNA levels and protein production. Moreover, it was also observed that recombinant protein yield was strongly dependent on the mRNA level. Therefore, three strategies aimed at increasing the amount of transgene mRNA were investigated. For the first approach, transfected cells were exposed to hypothermic conditions during the production phase. It was already known that lower temperatures increase protein production several fold in recombinant CHO DG44-derived cell lines. The second strategy involved the treatment of transfected cells with valproic acid, a histone deacetylase inhibitor that reduces the effects of gene silencing. The third approach aimed to increase transgene mRNA levels by overexpressing transcription factors and growth factors. With the first two strategies recombinant antibody yields of 60-80 mg/L were achieved whereas the untreated control transfections produced only 5-10 mg/L. Combination of the two strategies led to the production of 90 mg/L of antibody. Moreover, in the treated cultures, the steady-state level of transgene mRNA was 3-5 times higher than in the untreated cultures and remained stable up to 6 days post-transfection. Using the third approach, the increase in recombinant protein production was moderate and transgene mRNA amounts were only 2-fold higher in treated samples compared to the control. When specific proteins such as c-fos, c-jun, NF-kB, and acidic fibroblast growth factor (aFGF) were overexpressed the recombinant antibody production was 20 mg/L compared to 5 mg/L for the control transfection. Overexpression of either a transcription factor or a growth factor in combination with treatment with valproic acid allowed the recombinant protein yield to reach 90 mg/L. However, the benefit of the overexpressed factors was minimal compared to the effect of valproic acid alone. In conclusion, it was demonstrated that the level and stability of transgene mRNA are important factors for increasing volumetric yields in transiently transfected CHO DG44 cells. Furthermore, three approaches aimed to increase mRNA amounts were tested. Exposure to hypothermic conditions and treatment with valproic acid were the two best strategies tested. Both are simple, cost-effective, and scalable making transient gene expression in CHO DG44 cells a feasible alternative for rapid production of gram amounts of recombinant protein.
000135629 6531_ $$amammalian cell culture
000135629 6531_ $$arecombinant protein
000135629 6531_ $$atransient gene expression
000135629 6531_ $$amonoclonal antibody
000135629 6531_ $$ahypothermia
000135629 6531_ $$ahistone deacetylase
000135629 6531_ $$atranscription factor
000135629 6531_ $$agrowth factor
000135629 6531_ $$aculture de cellules de mammifère
000135629 6531_ $$aprotéine recombinante
000135629 6531_ $$aexpression génique transitoire
000135629 6531_ $$aanticorps monoclonal
000135629 6531_ $$ahypothermie
000135629 6531_ $$ahistone-deacetylase
000135629 6531_ $$afacteur de transcription
000135629 6531_ $$afacteur de croissance
000135629 700__ $$0240669$$aWulhfard, Sarah$$g169208
000135629 720_2 $$0240398$$aWurm, Florian$$edir.$$g107554
000135629 8564_ $$s1996139$$uhttps://infoscience.epfl.ch/record/135629/files/EPFL_TH4393.pdf$$yTexte intégral / Full text$$zTexte intégral / Full text
000135629 909C0 $$0252009$$pLBTC$$xU10180
000135629 909CO $$ooai:infoscience.tind.io:135629$$pthesis-bn2018$$pDOI2$$pDOI$$pthesis
000135629 918__ $$aSV$$cIBI1$$dEDBB
000135629 919__ $$aLBTC1
000135629 920__ $$b2009
000135629 970__ $$a4393/THESES
000135629 973__ $$aEPFL$$sPUBLISHED
000135629 980__ $$aTHESIS