Purification and characterization of three forms of differently glycosylated recombinant human granulocyte-macrophage colony-stimulating factor

Lee

Biological & Pharmaceutical Bulletin

et al. 2008

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) has been produced in several different cell types, and has different properties depending on the production process used. There has been no report of glycosylation ratio or of the role of glycans for plant cells-derived rhGM-CSF. We investigated the terminal sialylation of rhGM-CSF produced in rice cells (rrhGM-CSF) using lectins. Glycosylation ratios of rrhGM-CSF and yeast-derived rhGM-CSF (yrhGM-CSF) were evaluated by chemical deglycosylation. After intravenous administration to rats, the clearance rates of rrhGM-CSF and yrhGM-CSF as well as deglycosylated forms of them were evaluated. In vitro bioactivities of rrhGM-CSF and yrhGM-CSF were measured in dose-and time-dependent manners. Although rrhGM-CSF does not have terminal sialic acids, the glycosylation ratio was two-fold higher than that of yrhGM-CSF, and rrhGM-CSF sustained longer than yrhGM-CSF in the blood circulation. Moreover, yrhGM-CSF and rrhGM-CSF have almost same bioactivity via dose-and time-dependent manners. Deglycosylated rrhGM-CSF was cleared faster than native forms, while deglycosylated yrhGM-CSF and yrhGM-CSF were similarly cleared in blood circulation. These results suggest that the glycans on rrhGM-CSF are superior to the glycans on yrhGM-CSF in terms of in vivo stability.

Section: Proteinsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

The Glycosylation and in Vivo Stability of Human Granulocyte-Macrophage Colony-Stimulating Factor Produced in Rice Cells

Lee

Biological & Pharmaceutical Bulletin

et al. 2008

Overview of Protein Expression by Mammalian Cells

Gray¹

1997

CP Protein Science

This unit reviews the stages involved in protein production in mammalian cells using a stable-expression approach. Choice of cell type is discussed, as is transfection of the host cells, methods for selection and amplification of transformants, and growth of cells at appropriate scale for protein production. Since post-transcriptional modification and intracellular protein transportation are important features of recombinant-protein production in mammalian cells, some description of these mechanisms is included.

Biotech & Bioengineering

The effect of the dilution rate on CHO cell physiology and recombinant interferon‐γ production in glucose‐limited chemostat culture

Hayter

Curling

Gould

et al. 1993

The physiology of a recombinant Chinese hamster ovary cell line in glucose-limited chemostat culture was studied over a range of dilution rates (D = 0.008 to 0.20 h(-1)). The specific growth rate (micro) deviated from D at low dilution rates due to an increased specific death rate. Extrapolation of these data suggested a minimum specific growth rate of 0.011 h(-1) (micro(max) = 0.025 h(-1)) The metabolism at each steady state was characterized by determining the metabolic quotients for glucose, lactate, ammonia, amino acids, and interferon-gamma (IFN-gamma). The specific rate of glucose uptake increased linearly with mu, and the saturation constant for glucose (K(s)) was calculated to be 59.6 microM. There was a linear increase in the rate of lactate production with a higher yield of lactate from glucose at high growth rates. The decline in the rate of production of lactate, alanine, and serine at low growth rate was consistent with the limitation of the glycolytic pathway by glucose. The specific rate of IFN-gamma production increased with mu in a manner indicative of a growth-related product. Despite changes in the IFN-gamma production rate and cell physiology, the pattern of IFN-gamma glycosylation was similar at all except the lowest growth rates where there was increased production of nonglycosylated IFN-gamma.