Quantitative mapping of the N-linked sialyloligosaccharides of recombinant erythropoietin: Combination of direct high-performance anion-exchange chromatography and 2-aminopyridine derivatization

Rice, Kevin G.; Takahashi, Nobutaka; Namiki, Yoshihiro; Tran, An D.; Lisi, Peter J.; Lee, Yuan C.

doi:10.1016/0003-2697(92)90367-g

Cited by 60 publications

(23 citation statements)

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“…Furthermore, bisecting N-acetylglucosamine residues have never been detected in structural studies of the N-glycans of rEPOs and other glycoproteins synthesized in CHO cells. Figure 6 shows the N-glycan structures found in SpB, as well as other structures, which are known from previous studies (Rice et al, 1992;Watson et al, 1994;Hokke et al, 1995;Kanazawa et al, 1999) to be commonly present in rEPOs synthesized in CHO cells.…”

Section: N-glycan Profiles Of Reposmentioning

confidence: 89%

“…deduced from their elution positions on DEAE and Amide-80 chromatography, and from published data on the structures previously found to be present in rEPOs synthesized in CHO cells (Rice et al, 1992;Watson et al, 1994;Hokke et al, 1995;Kanazawa et al, 1999).…”

Section: N-glycan Profiles Of Reposmentioning

confidence: 99%

“…The structures of the oligosaccharides in individual Amide-80 HPLC fractions were then analysed by MALDI mass spectrometry to give the structures proposed in Table V. These structures were deduced from their molecular masses, their elution profiles on DEAE and Amide-80 HPLC, and from published data (Rice et al, 1992;Watson et al, 1994;Hokke et al, 1995;Kanazawa et al, 1999).…”

Section: N-glycan Profiles Of Reposmentioning

confidence: 99%

“…The elution positions of the various molecular species of the N-glycans present in these rEPOs have been allocated among the four regions of the Amide-80 chromatogram, which were defined by oligosaccharide standards with differing molecular masses, on the basis of their identification by MALDI mass Table V. Details of the mobile phases and elution gradients used for each of these HPLC systems were the same as those given in Fig 4. spectrometry in that fraction of SpB, or on the basis of the calculated molecular masses of molecular species of N-glycans identified in earlier studies (Rice et al, 1992;Watson et al, 1994;Hokke et al, 1995;Kanazawa et al, 1999) as commonly occurring in rEPOs synthesized in CHO cells. The relative contents of N-glycans in fraction Am-1 of these rEPOs (Fig 3, Table III) were found to be inversely correlated with their specific activities as estimated by in vivo bioassay (r ¼ ) 0AE898, P < 0AE005 ; Fig 7), and highly and significantly correlated with their specific activities as estimated by in vitro bioassay (r ¼ 0AE956, P < 0AE0005; Fig 8).…”

Section: N-glycan Profiles Of Reposmentioning

confidence: 99%

“…However, only limited data have been reported relating to the detailed N-glycan structures of the isoforms of EPO with their biological activities (Takeuchi et al, 1989). More detailed structural information about differences in glycosylation between EPOs can be obtained by N-glycan profiling techniques (Rice et al, 1992;Nimtz et al, 1993;Kanazawa et al, 1999;Skibeli et al, 2001), based on the release of N-glycans and their subsequent mapping. We have recently also described such a method for profiling the N-glycans of glycoproteins (Yuen et al, 2002), which can be applied to small (2-5 lg) amounts of EPO.…”

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Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

et al. 2003

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Summary. Eight preparations of recombinant human erythropoietin (EPO) with differing isoform compositions were produced by using different culture conditions and purification procedures. The N-glycan structures of these EPOs were analysed using a recently developed profiling procedure and identified using matrix-assisted laser desorption ionization mass spectrometry. The specific activities of each of the EPOs were estimated by in vivo and in vitro mouse bioassays. The eight EPOs were found to differ in their isoform compositions (as judged by isoelectric focusing), their N-glycan profiles, and in their in vivo and in vitro bioactivities. N-glycan analyses identified at least 23 different structures among these EPOs, including bi-, tri-and tetraantennary N-glycans, with or without fucosylation or N-acetyllactosamine extensions, and sialylated to varying degrees. Mass spectrometry also indicated the presence of N-glycans with incomplete outer chains, terminating in N-acetylglucosamine residues, and of molecular masses consistent with phosphorylated or sulphated oligomannoside structures. The tetrasialylated tetra-antennary N-glycan contents of the eight rEPOs were found to be significantly and positively correlated with their specific activities as estimated by mouse in vivo bioassay, and significantly and negatively correlated with their specific activities as estimated by mouse in vitro bioassay. It was concluded that the tetrasialylated tetra-antennary N-glycan content of EPO is a major determinant for its in vivo biological activity in the mouse.

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Section: N-glycan Profiles Of Reposmentioning

confidence: 89%

Section: N-glycan Profiles Of Reposmentioning

confidence: 99%

Section: N-glycan Profiles Of Reposmentioning

confidence: 99%

Section: N-glycan Profiles Of Reposmentioning

confidence: 99%

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Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

et al. 2003

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Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation

Yang

Butler²

2000

Biotechnol. Bioeng.

197

140

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The effect of ammonium chloride was determined on a culture of CHO cells transfected with the human erythropoietin (EPO) gene. Cell growth was inhibited above a culture concentration of 5 mM NH4Cl with an IC‐50 determined to be 33 mM. The specific production of EPO increased with the addition of NH4Cl above 5 mM. At 10 mM NH4Cl, the final cell density after 4 days in culture was significantly lower but the final yield of EPO was significantly higher. This appeared to be due to continued protein production after cell growth had ceased. The metabolic effects of added NH4Cl included higher specific consumption rates of glucose and glutamine and an increased rate of production of alanine, glycine, and glutamate. The EPO analyzed from control cultures had a molecular weight range of 33–39 kDa and an isoelectric point range of 4.06–4.67. Seven distinct isoforms of the molecule were identified by two‐dimensional electrophoresis. This molecular heterogeneity was ascribed to variable glycosylation. Complete enzymatic de‐glycosylation resulted in a single molecular form with a molecular mass of 18 kDa. Addition of NH4Cl to the cultures caused a significant increase in the heterogeneity of the glycoforms as shown by an increased molecular weight and pI range. Enzymatic de‐sialylation of the EPO from the ammonia‐treated and control cultures resulted in identical electrophoretic patterns. This indicated that the effect of ammonia was in the reduction of terminal sialylation of the glycan structures which accounted for the increased pI. Selective removal of the N‐glycan structures by PNGase F resulted in two bands identified as the O‐glycan linked structure (19 kDa) and the completely de‐glycosylated structure (18 kDa). The proportion of the O‐linked glycan structure was reduced, and its pI increased in cultures to which ammonia was added. Thus, the glycosylation pattern altered by the presence of ammonia included a reduction in terminal sialylation of all the glycans and a reduction in the content of the O‐linked glycan. The addition of a sialidase inhibitor to the cultures had no effect on the ammonia‐induced increase in EPO heterogeneity. Also, the effect of ammonia on glycosylation could not be mimicked using the weak base chloroquine in our system. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 370–380, 2000.

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The Endocrine System in Sports and Exercise

Kraemer¹,

Rogol²

2005

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This valuable new addition to the Encyclopaedia of Sports Medicine series provides a comprehensive and logical look at the principles and mechanisms of endocrinology as related to sports and exercise. It looks at growth hormone factors involved in exercise and the endocrinology of sport competition.It considers various factors and stresses on the body that may alter sporting performance. It covers topics from the acute responses and chronic adaptations of the human endocrine system to the muscular activity involved in conditioning exercise, physical labor, and sport activities.This book is an essential reference for helping to plan better programs of physical fitness, to prepare for sports competitions, and to manage the medical care of athletes.

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Quantitative mapping of the N-linked sialyloligosaccharides of recombinant erythropoietin: Combination of direct high-performance anion-exchange chromatography and 2-aminopyridine derivatization

Cited by 60 publications

References 27 publications

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

Effects of ammonia on CHO cell growth, erythropoietin production, and glycosylation

The Endocrine System in Sports and Exercise

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