Simple <i>in vivo</i> bioassay for erythropoietin

Kawamura, Akinori; Imai, Nobuo; Kawaguchi, Tsutomu; Hayakawa, Tetsuo

doi:10.1111/j.1365-2141.1991.tb08595.x

Cited by 16 publications

(6 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…O ensaio em camundongos policitêmicos baseia-se na incorporação de 59 Fe às células sangüíneas dos animais, previamente colocados sob pressão atmosférica reduzida. Esse procedimento utiliza radioisóto-pos, é de longa duração, e seu custo é elevado (4,6,(17)(18)(19). O bioensaio em camundongos normocitêmicos é executado usando animais normais e a atividade é avaliada pelo estímulo da produção de reticulócitos que correspondem ao último estágio da série eritróide da medula óssea, e são identificados através do retículo endoplasmático remanescente (6,9,16,18).…”

unclassified

Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

Schmidt

Ramos

Silva

et al. 2003

Arq Bras Endocrinol Metab

View full text Add to dashboard Cite

RESUMORealizou-se a identificação de eritropoietina humana recombinante em produtos farmacêuticos comerciais por eletroforese em gel de poliacrilamida e detecção com anticorpos específicos, demonstrando-se típi-ca banda larga, semelhante ao padrão de rhEPO da Farmacopéia Européia. Igualmente por focalização isoelétrica e imunodetecção, observaram-se 5-6 isoformas características de acordo com o laboratório produtor. A avaliação de potência efetuada através de ensaio biológico em camundongos normocitêmicos forneceu valores entre 67,6% e 119,4% em relação à declarada. A precisão dos ensaios combinados, calculada pela ponderação, variou de 200 a 389. Os testes de endotoxinas bacterianas, toxicidade e pH apresentaram valores variáveis conforme o lote. Concluiu-se destacando a importância dos testes e ensaios de controle para assegurar a qualidade lote a lote e garantir a eficácia terapêutica dos produtos farmacêuticos. The identification of rhEPO in pharmaceutical products was carried out by polyacrylamide gel electrophoresis and detection with specific antibodies that revealed a typical single broad band similar to that obtained with the European Pharmacopoeia Biological Reference Preparation for erythropoietin. The isoelectric focusing with immunodetection revealed extensive heterogeneity with 5-6 isoforms, characteristic according to the manufacturer. The potency was assessed by the normocythaemic mouse assay with values within 67.6% and 119.4% calculated against the stated potency. The precision evaluated by the weight was within 200 and 389, for the combined assays. The toxicity, bacterial endotoxins, and pH gave variable results according to the batch. In conclusion, we emphasize the importance of the batch-to-batch tests and assays that would guarantee the quality and therapeutic efficacy of the pharmaceutical products.

show abstract

unclassified

Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

Schmidt

Ramos

Silva

et al. 2003

Arq Bras Endocrinol Metab

View full text Add to dashboard Cite

show abstract

“…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: 53%

“…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%

See 1 more Smart Citation

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

Ramos

Schmidt

Andrade

et al. 2003

Braz J Med Biol Res

View full text Add to dashboard Cite

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

show abstract

“…Standard in-vivo bioassay of Epo. The method reported previously (Kawamura et a/, 1991;Hayakawa. et al 1992a) was adopted.…”

Section: Animalsmentioning

confidence: 99%

Characterization of haemolyser‐resistant cells increased in the blood of erythropoietin‐treated mice

et al. 1994

Self Cite

View full text Add to dashboard Cite

Recently, we reported a new in vivo assay method for erythropoietin (Epo) by means of counts of the number of haemolyser-resistant cells (HRCs) increased in Epo-injected mice. Here, we attempted to characterize the HRCs. Flow-cytometric studies revealed that HRCs obtained from Epo-injected mice expressed the transferrin receptor on their surface membranes. Furthermore, a fluorophotometric study suggested that the number of transferrin receptor-positive cells increased in a dose-dependent manner in response to treatment with Epo. On the other hand, flowcytometric and fluorophotometric studies of glycophorin A on HRCs using a rabbit antiglycophorin A antibody also showed a high expression of glycophorin A on them as compared with on HRCs from vehicle-treated animals (control). The results indicated that HRCs could be defined by their expression of both transferrin receptors and glycophorin A. We concluded that HRCs might be immature reticulocytes.

show abstract

Simple in vivo bioassay for erythropoietin

Cited by 16 publications

References 17 publications

Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

Avaliação da atividade e caracterização de eritropoietina humana recombinante em produtos farmacêuticos

Biological evaluation of recombinant human erythropoietin in pharmaceutical products

Characterization of haemolyser‐resistant cells increased in the blood of erythropoietin‐treated mice

Contact Info

Product

Resources

About