Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Morimoto, Kazushige; Tsuda, Eisuke; Said, Ahmed A.; Uchida, Eiji; Hatakeyama, Satoshi; Ueda, Masatsugu; Hayakawa, Tetsuo

doi:10.1007/bf01053197

Cited by 29 publications

(11 citation statements)

References 27 publications

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“…10,11) In the case of the fractionated sample with the same distribution pattern of asialo N-linked oligosaccharides, a linear relationship between in vivo bioactivity and sialic acid content was observed. 19) However, the low correlation between sialic acid content and in vivo bioactivity observed in the present study suggests the possibility that glycan moieties as well as sialic acid residues may have a significant impact on in vivo bioactivity of rhEPO subfractions. This is supported by previous reports indicating that the higher branching structure of N-glycan is required for in vivo bioactivity of EPO.…”

Section: )contrasting

confidence: 49%

Measurement of Sialic Acid Content Is Insufficient to Assess Bioactivity of Recombinant Human Erythropoietin

Yanagihara

Taniguchi

Hosono

et al. 2010

Biological & Pharmaceutical Bulletin

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To achieve quality control of therapeutic proteins, it is important to compare their biological potency with their clinical effects.1) Therefore, an animal-based assay is preferred for measuring their bioactivity. However, the use of laboratory animals poses ethical concerns, is time consuming and requires skilled staff and sophisticated experimental facilities. Furthermore, in vivo bioassays have low precision and accuracy, and hence, there is a need for an alternative assay method to estimate in vivo bioactivity.To date, attempts have been made to replace traditional bioassays with physicochemical techniques for potency determination of insulin and growth hormone because it is possible to design and validate manufacturing processes in the production of properly folded materials.2,3) Potency of these pharmaceutical preparations can be calculated using the content assay with HPLC, which quantifies the peak area of a product against that of a reference standard of known potency. The results of the HPLC assay have been shown to correlate well with those of the bioassay.2,3) In addition, the HPLC assay is rapid, precise and less labour intensive than the bioassay. However, for some glycoproteins, alternative assay methods using a quantitative charge-based separation technique, such as isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), have been shown to be feasible for estimating in vivo bioactivity 4,5) because negatively charged sialic acid residues are critical for in vivo bioactivity. For example, Mulders et al. 4) reported that quantitative assessment of the isoform distribution by IEF may provide a method for predicting in vivo biological potency of preparations containing recombinant human follicle-stimulating hormone (rhFSH). Close correlation between results of experimental in vivo bioactivities and IEF-predicted in vivo bioactivities suggest that the only factor affecting prediction of biological potency is the result of quantitative isoform distribution in rhFSH.Erythropoietin (EPO) is a glycoprotein hormone that regulates the red blood cell level by stimulating maturation of erythroid precursor cells. 6) Recombinant human EPO (rhEPO) produced in Chinese hamster ovary (CHO) cells is extensively used for serve anaemia therapy.7) EPO comprises a 165-amino acid protein with 40% of its molecular weight accounted for by carbohydrates.8,9) EPO contains three Nglycans located on Asn residues at positions 24, 38, and 83 and one mucin-type O-glycan located on the Ser-126 residue. The N-linked carbohydrates comprise bi-, tri-and tetraantennary oligosaccharides, which typically terminate with a negatively charged sialic acid residue. 10,11) Because of the variable number of sialic acid residues, the product of EPO usually exists as a mixture of isoforms with different isoelectric points. Several studies have reported that sialic acid residues are critical for in vivo bioactivity of EPO.12,13) Isoforms having a higher sialic acid content are seen to display higher in vivo bioactivity, longer seru...

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Section: )contrasting

confidence: 49%

Measurement of Sialic Acid Content Is Insufficient to Assess Bioactivity of Recombinant Human Erythropoietin

Yanagihara

Taniguchi

Hosono

et al. 2010

Biological & Pharmaceutical Bulletin

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“…There have also been studies of the relationships between the degree of sialylation of EPO and its in vivo and in vitro bioactivities. Thus in a number of studies, the degree of sialylation of EPO was found to be significantly and positively correlated with its in vivo bioactivity or its in vivo: in vitro bioactivity ratio (Imai et al, 1990;Morimoto et al, 1996;Storring et al, 1998). However, Takeuchi et al (1989), using the percentage exposure of the terminal galactose residues of the N-glycans of EPO as a measure of its sialylation, found no significant correlation between the degree of sialylation of EPO and its in vivo bioactivity.…”

Section: Discussionmentioning

confidence: 99%

“…However, Takeuchi et al (1989), using the percentage exposure of the terminal galactose residues of the N-glycans of EPO as a measure of its sialylation, found no significant correlation between the degree of sialylation of EPO and its in vivo bioactivity. The degree of sialylation of EPO has been reported to be significantly and negatively correlated with its in vitro bioactivity (Imai et al, 1990;Morimoto et al, 1996).…”

Section: Discussionmentioning

confidence: 99%

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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“…The heterogeneity arises largely through the variable N-glycan structures found at the three sites of attachment on the protein. The high sialic content of these glycans causes EPO to be a very acidic protein with reported pI values ranging from 3.0 to 4.7 (Davis et al, 1987;Imai et al, 1990;Morimoto et al, 1996;Schlags et al, 2002;Yang and Butler, 2000b).…”

Section: Discussionmentioning

confidence: 99%

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Restelli

Wang

Huzel

et al. 2006

Biotech & Bioengineering

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Human recombinant erythropoietin (rHuEPO) was produced from Chinese hamster ovary (CHO) cells transfected with the human EPO gene. The cells were grown in batch cultures in controlled bioreactors in which the set-points for dissolved oxygen varied between 3% and 200%. The cell-specific growth rate and final cell yield was significantly lower under hyperoxic conditions (200% DO). However, there was no significant difference in growth rates at other oxygen levels compared to control cultures run under a normoxic condition (50% DO). The specific productivity of EPO was significantly lower at a DO set-point of 3% and 200% but maintained a consistently high value between 10% to 100% DO. The EPO produced under all conditions as analyzed by two-dimensional electrophoresis showed a molecular weight range of 33 to 37 kDa and a low isoelectric point range of 3.5 to 5.0. This corresponds to a highly glycosylated and sialylated protein with a profile showing at least seven distinct isoforms. The glycan pattern of isolated samples of EPO was analyzed by weak anion exchange (WAX) HPLC and by normal-phase HPLC incorporating sequential digestion with exoglycosidase arrays. Assigned structures were confirmed by mass spectrometry (MALDI-MS). The most prominent glycan structures were core fucosylated tetranntenary with variable sialylation. However, significant biantennary, triantennary, and non-fucosylated glycans were also identified. Detailed analysis of these glycan structures produced under variable dissolved oxygen levels did not show consistently significant variations except for the ratio of fucosylated to non-fucosylated isoforms. Maximum core fucosylation (80%) was observed at 50% and 100% DO, whereas higher or lower DO levels resulted in reduced fucosylation. This observation of lower fucosylation at high or low DO levels is consistent with previous data reported for glycoprotein production in insect cells.

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Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Cited by 29 publications

References 27 publications

Measurement of Sialic Acid Content Is Insufficient to Assess Bioactivity of Recombinant Human Erythropoietin

Measurement of Sialic Acid Content Is Insufficient to Assess Bioactivity of Recombinant Human Erythropoietin

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

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

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