Simple In Vivo bioassay without radioisotopes for recombinant human erythropoietins

Hayakawa, Tetsuo; Wada, Michihito; Mizuno, Kimio; Abe, Satoshi; Miyashita, Masato; Ueda, Masatsugu

doi:10.1016/s1045-1056(05)80043-0

Cited by 12 publications

(11 citation statements)

References 19 publications

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“…The statistical requirements for assay validity, that is, significant regression and no significant deviation from linearity or parallelism of the log dose-log response lines (28), were fully met. The combination of two or three independent assays using a three-fold dose interval and eight animals per dose yielded lower confidence intervals than those reported in previous studies (14,16,17). This provides evidence that manual counts of reticulocytes can generate a valid assay with improved precision, albeit in a labor-intensive format.…”

Section: Discussionmentioning

confidence: 54%

“…The suitability of male or female mice has been investigated previously (3,13,15,17), but the results obtained with females in the present study were more precise and reproducible, with lower variability in the selected linear portion of the dose-response curve.…”

Section: Discussionmentioning

confidence: 60%

“…Reticulocyte numbers were also estimated by microscopy in a Neubauer cell chamber after treatment with a hemolyzing agent (selective hemolysis technique), by a procedure similar to that used in automated systems (14,17). The results obtained for the potency assessment of several rhEPO pharmaceutical preparations gave combined assay weights (N = 3) between 186 and 207.…”

Section: Discussionmentioning

confidence: 99%

“…The bioassay parameters were optimized by using the flow cytometry method for automated reticulocyte counting. Injection schedules and blood collection protocols were standardized in preliminary studies and consisted of the administration of a single, sc dose of EPO (from a concentration series with three-fold intervals) to mice aged 7 to 9 weeks, followed by blood sample collection after 96 h, or of multiple injections with blood collection after 24 h (14,16,17). The single injection assay with reticulocyte counting by the flow cytometry method was applied for the determination of the biopotency of six rhEPO pharmaceutical products from different manufacturers ( Table 2).…”

Section: Discussionmentioning

confidence: 99%

“…Although this bioassay is normally sensitive, precise and specific, the procedure has a number of drawbacks in that it requires the use of a radioisotope and elaborate animal preparation. 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: Discussionmentioning

confidence: 54%

Section: Discussionmentioning

confidence: 60%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

Ramos

Schmidt

Andrade

et al. 2003

Braz J Med Biol Res

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

et al. 1996

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Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol-1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between the in vivo bioactivity and the sialic acid content of the fractionated samples was observed until 12.1 mol mol-1 of EPO, there was no further increase in their activity over 12.4 mol mol-1 of EPO. On the other hand, an inverse relationship between the in vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that the in vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with alpha 2,6-sialyltransferase, but the in vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.

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In vitro bioassay for human erythropoietin based on proliferative stimulation of an erythroid cell line and analysis of carbohydrate-dependent microheterogeneity

Hammerling

Kroon

Wilhelmsen

et al. 1996

Journal of Pharmaceutical and Biomedical Analysis

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Simple In Vivo bioassay without radioisotopes for recombinant human erythropoietins

Cited by 12 publications

References 19 publications

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

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

In vitro bioassay for human erythropoietin based on proliferative stimulation of an erythroid cell line and analysis of carbohydrate-dependent microheterogeneity

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