title: Production of different glycosylation variants of the tumour-targeting mAb H10 in Nicotiana benthamiana: influence on expression yield and antibody degradation.
abstract: We previously described the expression of a tumour-targeting antibody (mAb H10) in Nicotiana benthamiana by vacuum-agro-infiltration and the remarkable yields of highly pure protein achieved. The objective of the present work was to investigate different strategies for transient overexpression of the mAb H10 in which glycan configuration was modulated and assess how these strategies affect the accumulation yield and stability of the antibody. To this aim, three procedures have been assayed: (1) Site-directed mutagenesis to abolish the glycosylation site; (2) endoplasmic reticulum retention (C-terminal SEKDEL fusion) to ensure predominantly high-mannose type glycans; and (3) expression in a N. benthamiana RNAi down-regulated line in which β1,2-xylosyltransferase and α1,3-fucosyltransferase gene expression is silenced. The three antibody variants (H10-Mut) (H10-SEKDEL) (H10(XylT/FucT)) were transiently expressed, purified and characterised for their glycosylation profile, expression/purification yield and antibody degradation pattern. Glycosylation analysis of H10(XylT/FucT) demonstrated the absence of plant complex-type sugars, while H10-SEKDEL, although substantially retained in the ER, revealed the presence of β1,2-xylose and α1,3-fucose residues, indicating a partial escape from the ER retrieval system. Antibody accumulation and purification yields were not enhanced by ER retention. All H10 antibody glyco-forms revealed greater degradation compared to the original, resulting mostly in the formation of Fab fragments. In the case of aglycosylated H10-Mut, more than 95% of the heavy chain was cleaved, confirming the pivotal role of the sugar moiety in protein stability. Identification of possible 'fragile' sites in the H10 antibody hinge region could be of general interest for the development of new strategies to reduce antibody degradation and increase the yield of intact IgGs in plants.
authors.affiliation: ENEA, Laboratorio Biotecnologie, UTBIORAD, C.R. Casaccia, Via Anguillarese 301, 00123, Rome, Italy. raffaele.lombardi@enea.it
authors.forename: Raffaele
authors.initials: R
authors.lastname: Lombardi
authors.forename: Marcello
authors.initials: M
authors.lastname: Donini
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Villani
authors.forename: Patrizia
authors.initials: P
authors.lastname: Brunetti
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kajiura
authors.forename: Matthew
authors.initials: M
authors.lastname: Paul
authors.forename: Julian K-C
authors.initials: JK
authors.lastname: Ma
authors.forename: Eugenio
authors.initials: E
authors.lastname: Benvenuto
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D000912:Antibodies, Neoplasm / Q000096:biosynthesis* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D016153:Genes, Suppressor; D006031:Glycosylation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis; D007074:Immunoglobulin G / Q000096:biosynthesis; D016297:Mutagenesis, Site-Directed; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D015202:Protein Engineering; D055550:Protein Stability; D011523:Protoplasts / Q000378:metabolism; D034622:RNA Interference; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D011134:Polysaccharides