Hills A E, Patel A, Boyd P, James D C
Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
Biotechnol Bioeng. 2001 Oct 20;75(2):239-51. doi: 10.1002/bit.10022.
Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.
重组治疗性免疫球蛋白G（IgG）分子Fc区域Asn（297）处的可变N-糖基化，特别是末端半乳糖基化和唾液酸化，可能会影响重组治疗性抗体的药代动力学行为和效应功能。我们研究了IgG Fc糖基化可通过操纵细胞核苷酸糖代谢来控制的假说。在对照培养物中，GS-NS0细胞在培养过程中产生的重组人源化IgG1的Fc结构域相关的N-聚糖主要是双天线型，具有可变的β-半乳糖基化（平均0.3摩尔半乳糖复合N-聚糖（-1））结构，没有明显的平分N-乙酰葡糖胺残基、唾液酸化或α1,3-连接的半乳糖基化。然而，存在可变比例（5%至15%）的高甘露糖（Man5至Man9）寡糖。为了操纵半乳糖基化所需的核苷酸糖前体UDP-Gal的细胞含量，我们在培养基中加入了10 mM葡糖胺或10 mM半乳糖。在前一种情况下，观察到细胞UDP-N-乙酰己糖胺含量增加了17倍，同时总UDP-己糖减少（33%），尽管UDP-Glc:UDP-Gal的比例（4:1）没有变化。与细胞UDP-糖含量的这些变化相关的是Fc衍生寡糖的半乳糖基化显著降低（57%）。Asn（297）处高甘露糖型N-聚糖（特别是Man5，N-乙酰葡糖胺基转移酶I的底物）的比例不受影响。相反，在培养物中加入10 mM半乳糖可特异性刺激UDP-Gal含量增加近五倍。然而，这仅导致Fc N-聚糖的β1,4-半乳糖基化有最小的、不显著的增加（6%）。即使细胞CMP-唾液酸（CMP-SA）含量增加了44倍，加入CMP-唾液酸（CMP-SA）前体N-乙酰甘露糖胺（20 mM）后唾液酸化也没有改善。分批培养过程中重组IgG1 Fc糖基化的分析表明，β1,4-连接的半乳糖基化在培养过程中略有下降，尽管在培养后期，裂解细胞释放的蛋白酶和糖苷酶可能导致半乳糖基化更显著的下降。这些数据表明：（i）空间位阻对Fc N-聚糖加工的影响；（ii）细胞核苷酸糖含量的变化可能影响Fc N-聚糖加工的程度；（iii）Fc N-糖基化直接代谢控制的潜力。
