Herman Xavier, Far Johann, Courtoy Adeline, Bouhon Laurent, Quinton Loïc, De Pauw Edwin, Chaumont François, Navarre Catherine
Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium.
Mass Spectrometry Laboratory-MolSys, GIGA Proteomics Facility, University of Liège, Liège, Belgium.
Front Plant Sci. 2021 Jan 27;12:634023. doi: 10.3389/fpls.2021.634023. eCollection 2021.
Bright Yellow-2 (BY-2) suspension cells are among the most commonly used plant cell lines for producing biopharmaceutical glycoproteins. Recombinant glycoproteins are usually produced with a mix of high-mannose and complex -glycans. However, -glycan heterogeneity is a concern for the production of therapeutic or vaccine glycoproteins because it can alter protein activity and might lead to batch-to-batch variability. In this report, a BY-2 cell line producing glycoproteins devoid of complex -glycans was obtained using CRISPR/Cas9 edition of two genes, whose activity is a prerequisite for the formation of all complex -glycans. The suppression of complex -glycans in the -knocked out (KO) cell lines was assessed by Western blotting. Lack of β1,2-xylose residues confirmed the abolition of GnTI activity. Unexpectedly, α1,3-fucose residues were still detected albeit dramatically reduced as compared with wild-type cells. To suppress the remaining α1,3-fucose residues, a second genome editing targeted both and α () genes. No β1,2-xylose nor α1,3-fucose residues were detected on the glycoproteins produced by the -KO cell lines. Absence of complex -glycans on secreted glycoproteins of -KO and -KO cell lines was confirmed by mass spectrometry. Both cell lines produced high-mannose -glycans, mainly Man5 (80 and 86%, respectively) and Man4 (16 and 11%, respectively). The high degree of -glycan homogeneity and the high-mannose -glycosylation profile of these BY-2 cell lines is an asset for their use as expression platforms.
明亮黄-2（BY-2）悬浮细胞是生产生物制药糖蛋白最常用的植物细胞系之一。重组糖蛋白通常是高甘露糖型和复合型聚糖的混合物。然而，聚糖异质性是治疗性或疫苗糖蛋白生产中的一个问题，因为它会改变蛋白质活性，并可能导致批次间的差异。在本报告中，通过CRISPR/Cas9编辑两个基因获得了一个产生不含复合型聚糖的糖蛋白的BY-2细胞系，这两个基因的活性是所有复合型聚糖形成的先决条件。通过蛋白质免疫印迹法评估敲除（KO）细胞系中复合型聚糖的抑制情况。缺乏β1,2-木糖残基证实了GnTI活性的消除。出乎意料的是，与野生型细胞相比，尽管α1,3-岩藻糖残基显著减少，但仍能检测到。为了抑制剩余的α1,3-岩藻糖残基，第二次基因组编辑靶向了和α（）基因。在-KO细胞系产生的糖蛋白上未检测到β1,2-木糖和α1,3-岩藻糖残基。通过质谱法证实-KO和-KO细胞系分泌的糖蛋白上没有复合型聚糖。这两种细胞系都产生了高甘露糖型聚糖，主要是Man5（分别为80%和86%）和Man4（分别为16%和11%）。这些BY-2细胞系高度的聚糖同质性和高甘露糖型糖基化谱是其作为表达平台的优势。
