A genome‐edited N. benthamiana line for industrial‐scale production of recombinant glycoproteins with targeted N‐glycosylation

Abstract

Control over glycosylation is an important quality parameter in recombinant protein production. Here, we demonstrate the generation of a marker‐free genome edited Nicotiana benthamiana N‐glycosylation mutant (NbXF‐KO) carrying inactivated β1,2‐xylosyltransferase and α1,3‐fucosyltransferase genes. The knockout of seven genes and their stable inheritance was confirmed by DNA sequencing. Mass spectrometric analyses showed the synthesis of N‐glycans devoid of plant‐specific β1,2‐xylose and core α 1,3‐fucose on endogenous proteins and a series of recombinantly expressed glycoproteins with different complexities. Further transient glycan engineering towards more diverse human‐type N‐glycans resulted in the production of recombinant proteins decorated with β1,4‐galactosylated and α2,6‐sialylated structures, respectively. Notably, a monoclonal antibody expressed in the NbXF‐KO displayed glycosylation‐dependent activities. Collectively, the engineered plants grow normally and are well suited for upscaling, thereby meeting industrial and regulatory requirements for the production of high‐quality therapeutic proteins.

Efficient Expression of Functionally Active Aflibercept with Designed N-glycans

“…A magnICON ® vector carrying an aflibercept DNA fragment (pICH26211α-aflibercept, Figure S2A ) was delivered through agroinfiltration to leaf cells of a glycoengineered N. benthamiana that synthesizes complex N-glycans, lacking plant-specific xylose and fucose (GnGn structures) [ 18 , 24 ]. Aflibercept expression was monitored at 4 to 5 dpi in total soluble protein extracts.…”

“…The majority of N-glycans are core-fucosylated. Glycoengineered plants were used in this study since they usually generate antibodies with largely homogeneous and reproducible GnGn structures, lacking plant-specific sugar residues [ 18 , 24 , 31 ]. Moreover, glycoengineered plant-derived IgGs often exhibit increased effector functions compared to orthologues produced in CHO cells or in wild-type plants [ 32 , 33 ].…”

“…Glycoengineered N. benthamiana plants [ 18 , 24 ] were grown in a growth chamber under controlled conditions at 24 °C, 60% humidity, with a 16 h light/8 h dark photoperiod.…”

“… N-glycan composition of aflibercept expressed in N. benthamiana plants deficient in core xylose and fucose [ 18 , 24 ]. Four variants were analyzed by LC-ESI-MS: AFLI GnGn and AFLI Sia purified from TSP and IF.…”

Implications of O-glycan modifications in the hinge region of a plant-produced SARS-CoV-2-IgA antibody on functionality

“…Their endogenous N- and O-glycosylation patterns differ from humans, which has hampered the broad application of the expression system to produce human therapeutics ( Strasser et al, 2021 ). While there has been considerable effort in glycoengineering popular expression hosts such as Nicotiana benthamiana , N. tabacum , tobacco BY2 cells and the moss Physcomitrella patens towards humanized- and customized N-glycosylation ( Bakker et al, 2001 ; Koprivova et al, 2004 ; Strasser et al, 2008 ; Kallolimath et al, 2016 ; Limkul et al, 2016 ; Mercx et al, 2017 ; Jansing et al, 2019 ; Bohlender et al, 2020 ; Herman et al, 2021 ; Göritzer et al, 2022 ; Kogelmann et al, 2023 ) limited attention was cast towards modulating the plant endogenous O-glycosylation pathway ( Castilho et al, 2012 ; Yang et al, 2012 ; Parsons et al, 2013 ; Dicker et al, 2016 ; Ramírez-Alanis et al, 2018 ; Mócsai et al, 2021 ; Uetz et al, 2022 ). Plant-endogenous HyP and further modifications with pentoses (arabinoses) were found in several recombinantly produced proteins such as IgA1, MUC1, EPO-Fc, and Ara h 2 ( Karnoup et al, 2005 ; Weise et al, 2007 ; Pinkhasov et al, 2011 ; Castilho et al, 2012 ; Yang et al, 2012 ; Üzülmez et al, 2021 ).…”