Control of rHuEPO biological activity: The role of carbohydrate

Elliott, Steve; Egrie, Joan C.; Browne, Jeff; Lorenzini, Tony; Busse, Leigh; Rogers, Norma; Ponting, I. L. O.

doi:10.1016/j.exphem.2004.08.004

Cited by 177 publications

(152 citation statements)

References 49 publications

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“…Removal of two chains resulted in about a 50-fold decrease. Overall, there was a general, positive correlation between number of carbohydrate chains and in-vivo-specific activity [39] (Fig. 3).…”

Section: Role Of Carbohydrate On Epo Activitymentioning

confidence: 99%

“…2 Serum clearance is the primary determinant of in vivo activity of erythropoietin isoform (from [42], with permission) would recognize the new carbohydrate attachment points in rHuEPO. Using in vitro mutagenesis, rHuEPO glycosylation analogs with varying numbers of N-linked carbohydrate chains were successfully constructed and became available for testing [11,39]. Addition of one new carbohydrate chain increased approximately 1.6-fold the in-vivo-specific activity in rodents, and addition of two chains resulted in greater than twofold increase, as measured by the exhypoxic polycythemic mouse bioassay [39].…”

Section: Role Of Carbohydrate On Epo Activitymentioning

confidence: 99%

“…Attached carbohydrate on rHuEpo comprises approximately 40% of the mass of the molecule [7]. Whereas differences in carbohydrate content of EPO can affect affinity to EPOR, carbohydrate also plays an important role in maintaining the molecule in circulation (clearance) [39]. In addition to its effect on clearance, the increased content of negatively charged carbohydrates can also increase solubility and decrease adhesive properties of protein.…”

Section: Role Of Carbohydrate On Epo Activitymentioning

confidence: 99%

“…Using in vitro mutagenesis, rHuEPO glycosylation analogs with varying numbers of N-linked carbohydrate chains were successfully constructed and became available for testing [11,39]. Addition of one new carbohydrate chain increased approximately 1.6-fold the in-vivo-specific activity in rodents, and addition of two chains resulted in greater than twofold increase, as measured by the exhypoxic polycythemic mouse bioassay [39]. In contrast, when one N-linked chain was removed from the protein backbone of rHuEPO, the in-vivo-specific activity decreased approximately two-to fourfold.…”

Section: Role Of Carbohydrate On Epo Activitymentioning

confidence: 99%

See 3 more Smart Citations

Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact

Kiss¹,

Elliott

Jedynasty³

et al. 2010

Eur J Clin Pharmacol

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Cloning of the human erythropoietin (EPO) gene and development of the first recombinant human erythropoietin (rHuEPO) drug were truly breakthroughs. This allowed a deeper understanding of the structure and pharmacology of rHuEpo, which in turn inspired the discovery and development of additional erythropoiesisstimulating agents (ESAs). In vivo specific activity and serum half-life of rHuEPO are influenced by the amount and structure of the attached carbohydrate. Increased numbers of sialic acids on carbohydrate attached to rHuEPO correlated with a relative increase in in-vivospecific activity and increased serum half-life. The effect of increasing the number of sialic-acid-containing carbohydrates on in-vivo-specific activity was explored. Initial research focused on solving the problem of how the protein backbone could be engineered so a cell would add more carbohydrate to it. Additional work resulted in darbepoetin alfa, a longer-acting molecule with two additional carbohydrate chains.

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Section: Role Of Carbohydrate On Epo Activitymentioning

confidence: 99%

Section: Role Of Carbohydrate On Epo Activitymentioning

confidence: 99%

Section: Role Of Carbohydrate On Epo Activitymentioning

confidence: 99%

Section: Role Of Carbohydrate On Epo Activitymentioning

confidence: 99%

See 2 more Smart Citations

Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact

Kiss¹,

Elliott

Jedynasty³

et al. 2010

Eur J Clin Pharmacol

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show abstract

“…N-glycosylation has widely been reported to exert significant effects on several properties of glycoproteins, including antigenicity, solubility, protein folding, protease resistance, pharmacokinetics or biological activity (Schauer 1988;Kobata 1992;Yusa et al 2005). Moreover, sialylation is known to be able to influence the in vivo circulatory lifetime of these proteins, which is crucial to their therapeutic value (Dordal et al 1985;Takeuchi et al 1989;Imai et al 1990;Misaizu et al 1995;Koury 2003;Yuen et al 2003;Elliott et al 2004).…”

Section: Introductionmentioning

confidence: 99%

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells

et al. 2006

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The necessity to perform serum-free cultures to produce recombinant glycoproteins generally requires an adaptation procedure of the cell line to new environmental conditions, which may therefore induce quantitative and qualitative effects on the product, particularly on its glycosylation. In previous studies, desialylation of EPO produced by CHO cells was shown to be dependent on the presence of serum in the medium. In this paper, to discriminate between the effects of the adaptation procedure to serum-free medium and the effects of the absence of serum on EPO production and glycosylation, adapted and nonadapted CHO cells were grown in serum-free and serum-containing media. The main kinetics of CHO cells were determined over batch processes as well as the glycosylation patterns of produced EPO by HPCE-LIF. A reversible decrease in EPO production was observed when cells were adapted to SFX-CHO TM medium, as the same cells partially recovered their production capacity when cultivated in serum-containing medium or in the enriched SFM TM serum-free medium. More interestingly, EPO desialylation that was not observed in both serum-free media was restored if the serum-independent cells were recultured in presence of serum. In the same way, while the serum-independent cells did not release a sialidase activity in both serum-free media, a significant activity was recovered when serum was added. In fact, the cell adaptation process to serum-free conditions did not specifically affect the sialidase release and the cellular mechanism of protein desialylation, which appeared to be mainly related to the presence of serum for both adapted and non-adapted cells.

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Animal Cells, Hybridomas, Human Antibody Production

Warren¹,

Subramanian²

2010

Encyclopedia of Industrial Biotechnology

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Cellular response to perturbations in the microenvironment manifests as a surge of physiological changes that have great impact on cellular metabolism and propagation. In bioprocess systems, mammalian cells engineered for the production of monoclonal antibodies or therapeutic proteins are sensitive to fluctuations in the culture environment. These fluctuations often directly translate to changes in maximum attainable cell concentration and/or specific protein productivity. In conjunction with feeding strategy optimization, the ability to understand and optimize the physical environment of animal culture systems is key to achieving a process that delivers consistent production of high titer batches. In this article we have reviewed comprehensively two critical physical culture parameters: osmolality and temperature, for their effects on general cellular physiology as well as their impacts on overall culture performance in bioprocess systems. Extreme levels of osmolality lead to volume‐regulatory responses, cytoskeletal reorganization, and trigger distinct cellular signaling pathways. Cellular responses to hypothermic conditions include reduced metabolic and growth rates, reduction of apoptosis frequency, and a cold‐shock response which involves the increased synthesis of several proteins. Exposure of cells to hyperosmotic conditions and/or subphysiological temperature (32–35°C) has been shown to enhance specific protein productivity in bioprocess systems. As live viruses are also commonly produced in industrial cell culture systems for vaccine processes, we have extended the scope of this article to include the effects of fluctuations in culture osmolality and temperature on the production of live viruses.

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Control of rHuEPO biological activity: The role of carbohydrate

Cited by 177 publications

References 49 publications

Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact

Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs), including the hyperglycosylated ESA, darbepoetin alfa: an update of the rationale and clinical impact

Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells

Animal Cells, Hybridomas, Human Antibody Production

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