Inactivation of and Genes in BY-2 Cells Results in Glycoproteins With Highly Homogeneous, High-Mannose -Glycans

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.