The International Standard for Recombinant DNA-derived Erythropoietin: collaborative study of four recombinant DNA-derived erythropoietins and two highly purified human urinary erythropoietins

Storring, P. L.; Das, Rose E. Gaines

doi:10.1677/joe.0.1340459

Cited by 82 publications

(46 citation statements)

References 35 publications

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“…Combinations of two or three independent bioassays gave potencies and confidence limits for the preparations that satisfied the specifications of the European Pharmacopoeia (15) and gave a precision that agreed with previously published values (13,19). However, by comparing the results and precision of the single injection assay with those of the multiple daily injection assay it can be seen that for the single injection at least two assays were generally necessary to achieve acceptable limits, whereas for the multiple injection assay the confidence intervals were lower, and the precision and reproducibility were higher using results from only one assay.…”

Section: Discussionsupporting

confidence: 83%

“…Currently, the normocythemic mouse bioassay is performed in normal animals with single (13)(14)(15) or multiple injections (16)(17)(18) and the biological activity of rhEPO is measured by the stimulation of reticulocyte production (3,(16)(17)(18). Bioassays, in particular that involving a single injection into normocythemic mice, have been widely applied in standardization studies and in the potency evaluation of pharmaceutical preparations (13,15,19); however, although they are usually robust, some improvement is needed with regard to response variability and accuracy, most importantly when assessing the potency of commercial rhEPO batches from various sources (13,14).…”

Section: Introductionmentioning

confidence: 99%

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Biological evaluation of recombinant human erythropoietin in pharmaceutical products

Ramos

Schmidt

Andrade

et al. 2003

Braz J Med Biol Res

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The potencies of mammalian cell-derived recombinant human erythropoietin pharmaceutical preparations, from a total of five manufacturers, were assessed by in vivo bioassay using standardized protocols. Eight-week-old normocythemic mice received a single subcutaneous injection followed by blood sampling 96 h later or multiple daily injections with blood sampling 24 h after the last injection. Reticulocyte counting by microscopic examination was employed as the endpoint using the brilliant cresyl blue or selective hemolysis methods, together with automated flow cytometry. Different injection schedules were investigated and dose-response curves for the European Pharmacopoeia Biological Reference Preparation of erythropoietin were compared. Manual and automated methods of reticulocyte counting were correlated with respect to assay validity and precision. Using 8 mice per treatment group, intra-assay precision determined for all of the assays in the study showed coefficients of variation of 12.1-28.4% for the brilliant cresyl blue method, 14.1-30.8% for the selective hemolysis method and 8.5-19.7% for the flow cytometry method. Applying the single injection protocol, a combination of at least two independent assays was required to achieve the precision potency and confidence limits indicated by the manufacturers, while the multiple daily injection protocol yielded the same acceptable results within a single assay. Although the latter protocol using flow cytometry for reticulocyte counting gave more precise and reproducible results (intra-assay coefficients of variation: 5.9-14.2%), the well-characterized manual methods provide equally valid alternatives for the quality control of recombinant human erythropoietin therapeutic products. Correspondence

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Section: Discussionsupporting

confidence: 83%

Section: Introductionmentioning

confidence: 99%

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

Ramos

Schmidt

Andrade

et al. 2003

Braz J Med Biol Res

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“…The in vivo post-hypoxic polycythaemic mouse bioassay (Cotes & Bangham, 1961) was carried out with modifications, as described previously (Storring et al, 1998). All bioassay estimates were in terms of the International Standard for Recombinant DNA-derived Erythropoietin (IS) (Storring & Gaines Das, 1992).…”

Section: Bioassaysmentioning

confidence: 99%

“…In the case of rEPO, the isoform composition is influenced by the cell line and culture conditions used for its synthesis, and the selectivity of the purification procedures used to isolate the preparation. Thus differences have been found between the isoforms of EPOs synthesized in different cell lines, between different products synthesized in the same type of cell line and even between batches of the same product (Sasaki et al, 1987;Wide & Bengtsson, 1990; Rice et al,EPOs differing in their isoform composition have been found to differ in their biological activities (Fukuda et al, 1989;Takeuchi et al, 1989;Storring & Gaines Das, 1992;Storring et al, 1998). However, structure-activity studies among the isoforms of EPO have usually used only chargebased separations by isoelectric focusing (IEF) or electrophoresis to distinguish between the isoform compositions of different EPOs, ascribing observed differences between isoforms mainly to differences in sialylation.…”

mentioning

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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“…It contains both N-linked and O-linked carbohydrates that have been structurally determined (Sasaki et al, 1988). Chinese hamster ovary cell-derived recombinant HuEPO contains similar carbohydrate moieties to that of human urinary EPO, although the carbohydrate on CHO-EPO and other recombinantly produced EPO is different f r o m that of urinary EPO (Storring & Das, 1992). In contrast, the gene product ex-pressed in Escherichia coli (E. coli EPO) is nonglycosylated and has low biological activity in vivo.…”

mentioning

confidence: 99%

Recombinant human erythropoietin (rHuEPO): Cross‐linking with disuccinimidyl esters and identification of the interfacing domains in EPO

et al. 1993

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Several amino groups of recombinant human erythropoietin are selectively cross-linked by specific cross-linkers including disuccinimidyl suberate or dithiobis(succinimidy1 propionate). Intramolecular cross-linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the crosslinkers, which possess an I1-12-A length of a spacer between two reacting groups. Intramolecularly cross-linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 A in the solution state. These interfacing amino groups include Lys 20-Lys 154, Lys 45-Lys 140, Lys 52-Lys 154, Lys 52-Lys 140, and Ala 1-Lys 116. A comparison of the cross-linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation. These results fit a structural model similar to that of human growth hormone, in which four a-helical bundles and a long stretch of &sheet structure are involved in the active protein.

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The International Standard for Recombinant DNA-derived Erythropoietin: collaborative study of four recombinant DNA-derived erythropoietins and two highly purified human urinary erythropoietins

Cited by 82 publications

References 35 publications

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

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

Recombinant human erythropoietin (rHuEPO): Cross‐linking with disuccinimidyl esters and identification of the interfacing domains in EPO

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