Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody 英文参考文献.docVIP

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Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody 英文参考文献.doc

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Rapid High Yield Production of Different Glycoforms of Ebola Virus Monoclonal Antibody 英文参考文献

RapidHighYieldProductionofDifferentGlycoformsof EbolaVirusMonoclonalAntibody AlexandraCastilho1,NatashaBohorova2,JosephineGrass3,OgnianBohorov2,LarryZeitlin2 ,Kevin Whaley2,FriedrichAltmann3,HertaSteinkellner1* 1DepartmentofAppliedGeneticsandCellBiology,UniversityofNaturalResourcesandLifeSciences,Vienna,Austria,2MappBiopharmaceutical,SanDiego,California, UnitedStatesofAmerica,3DepartmentofChemistry,UniversityofNaturalResourcesandLifeSciences,Vienna,Austria Abstract Background: Fc-glycosylation of monoclonal antibodies (mAbs) has profound implications on the Fc-mediated effector functions.Alterationofthisglycosylationmayaffecttheefficiencyofanantibody.However,difficultiesintheproductionof mAbswithhomogeneousN-glycosylationprofilesinsufficientamountshamperinvestigationsofthepotentialbiological impactofdifferentglycanresidues. Methodology/PrincipalFindings:Herewesetouttoevaluateatransientplantviralbasedproductionsystemfortherapid generation of different glycoforms of a monoclonal antibody. Ebola virus mAb h-13F6 was generated using magnICON expression system in Nicotiana benthamiana, a plant species developed for commercial scale production of therapeutic proteins.h-13F6wasco-expressedwithaseriesofmodifiedmammalianenzymesinvolvedintheprocessingofcomplexN- glycans. Using wild type (WT) plants and the glycosylation mutant DXTFT that synthesizes human like biantennary N- glycans with terminal N-acetylglucosamine on each branch (GnGn structures) as expression hosts we demonstrate the generationofh-13F6complexN-glycanswith(i)bisectedstructures,(ii)corea1,6fucosylationand(iii)b1,4galactosylated oligosaccharides.InadditionweemphasizethesignificanceofprecisesubGolgilocalizationofenzymesforengineeringof IgGFc-glycosylation. Conclusion:Themethoddescribed hereallowstheefficientgenerationofaseriesofdifferenthuman-likeglycoforms at large homogeneity of virtually any antibody within one week after cDNA delivery to plants. This accelerates follow up functionalstudies andthus