Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N‐glycosylation of human secreted alkaline phosphatase

Abstract

We report for the first time that culture conditions, specifically culture medium supplementation with nucleotide-sugar precursors, can alter significantly the N-linked glycosylation of a recombinant protein in plant cell culture. Human secreted alkaline phosphatase produced in tobacco NT1 cell suspension cultures was used as a model system. Plant cell cultures were supplemented with ammonia (30 mM), galactose (1 mM) and glucosamine (10 mM) to improve the extent of N-linked glycosylation. The highest levels of cell density and active extracellular SEAP in supplemented cultures were on average 260 g/L and 0.21 U/mL, respectively, compared to 340 g/L and 0.4 U/mL in unsupplemented cultures. The glycosylation profile of SEAP produced in supplemented cultures was determined via electrospray ionization mass spectrometry with precursor ion scanning and compared to that of SEAP produced in unsupplemented cultures. In supplemented and unsupplemented cultures, two biantennary complex-type structures terminated with one or two N-acetylglucosamines and one paucimannosidic glycan structure comprised about 85% of the SEAP glycan pool. These three structures contained plant-specific xylose and fucose residues and their relative abundances were affected by each supplement. High mannose structures (6-9 mannose residues) accounted for the remaining 15% glycans in all cases. The highest proportion (approximately 66%) of a single complex-type biantennary glycan structure terminated in both antennae by N- acetylglucosamine was obtained with glucosamine supplementation versus only 6% in unsupplemented medium. This structure is amenable for in vitro modification to yield a more human-like glycan and could serve as a route to plant cell culture produced therapeutic glycoproteins.

Glycan structure and serum half-life of recombinant CTLA4Ig, an immunosuppressive agent, expressed in suspension-cultured rice cells with coexpression of human β1,4-galactosyltransferase and human CTLA4Ig

“…In addition, it is reported that change of the ammonium concentration during the culture affects the amount of Gal and GlcNAc residues in CHO cells [43]. In plants, glycosylation is also altered by supplementing glucosamine and ammonia into the culture medium [44]. Further study of the increases in galactosylated glycans is required.…”

Glycoform Modification of Secreted Recombinant Glycoproteins through Kifunensine Addition during Transient Vacuum Agroinfiltration

“…Thus, if these plant-specific glycans are present in an injected pharmaceutical product, the glycoprotein could trigger immunological response, or at least, result in a short circulation half-life. N -glycans of proteins produced from mammals are often terminated in β(1,4)-galactose and sialic acid; sialic acid is particularly important as it typically increases the circulation half-life of proteins [ 5 ].…”

Production of recombinant human granulocyte macrophage‐colony stimulating factor in rice cell suspension culture with a human‐like N‐glycan structure

“…, 2001; Joosten and Shuler, 2003; Yoon et al. , 2004; Becerra‐Arteaga and Shuler, 2007). Moreover, high‐mannose type N ‐glycans were detected only in intracellular glycoproteins in rice suspension‐cultured cells (Maeda and Kimura, 2006), suggesting that the high‐mannose type glycans exist only on the rice intracellular glycoproteins.…”