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1.
Glycobiology ; 13(4): 305-13, 2003 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-12626384

RESUMEN

Fabry disease is a lysosomal storage disease arising from deficiency of the enzyme alpha-galactosidase A. Two recombinant protein therapeutics, Fabrazyme (agalsidase beta) and Replagal (agalsidase alfa), have been approved in Europe as enzyme replacement therapies for Fabry disease. Both contain the same human enzyme, alpha-galactosidase A, but they are produced using different protein expression systems and have been approved for administration at different doses. To determine if there is recognizable biochemical basis for the different doses, we performed a comparison of the two drugs, focusing on factors that are likely to influence biological activity and availability. The two drugs have similar glycosylation, both in the type and location of the oligosaccharide structures present. Differences in glycosylation were mainly limited to the levels of sialic acid and mannose-6-phosphate present, with Fabrazyme having a higher percentage of fully sialylated oligosaccharides and a higher level of phosphorylation. The higher levels of phosphorylated oligomannose residues correlated with increased binding to mannose-6-phosphate receptors and uptake into Fabry fibroblasts in vitro. Biodistribution studies in a mouse model of Fabry disease showed similar organ uptake. Likewise, antigenicity studies using antisera from Fabry patients demonstrated that both drugs were indistinguishable in terms of antibody cross-reactivity. Based on these studies and present knowledge regarding the influence of glycosylation on protein biodistribution and cellular uptake, the two protein preparations appear to be functionally indistinguishable. Therefore, the data from these studies provide no rationale for the use of these proteins at different therapeutic doses.


Asunto(s)
Isoenzimas/farmacocinética , alfa-Galactosidasa/farmacocinética , Animales , Sitios de Unión , Unión Competitiva , Línea Celular , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Enfermedad de Fabry/tratamiento farmacológico , Enfermedad de Fabry/enzimología , Humanos , Inmunohistoquímica , Isoenzimas/genética , Isoenzimas/farmacología , Cinética , Ratones , Ratones Noqueados , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Espectrometría de Masa por Ionización de Electrospray , Factores de Tiempo , Distribución Tisular , alfa-Galactosidasa/genética , alfa-Galactosidasa/farmacología
2.
Biologicals ; 30(3): 245-54, 2002 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-12217348

RESUMEN

Thyroid stimulating hormone (TSH), a pituitary glycoprotein hormone, is a potent inducer of intracellular cAMP production. Two methods for measuring TSH bioactivity were evaluated and compared. One assay is based on using a radioimmunoassay (RIA) to measure the recombinant human TSH-induced increase in cAMP using a bovine thyroid membrane isolate. The other is based on a Chinese hamster ovary (CHO) cell line that has been transfected with the TSH receptor and a cAMP-responsive luciferase reporter. The within-assay coefficient of variation for the membrane-based assay was determined to be approximately 35% compared with approximately 25% for the cell-based assay. Twenty-one preparations of recombinant human TSH (rhTSH) were tested using both methods. No significant difference was detected between the data sets and no assay bias was present. Both assay systems provide a suitable means for measuring the activity of rhTSH. The advantage of the membrane-based assay is the relatively small quantity of TSH needed for analysis. However, the average time required to analyse a sample using the membrane-based method was more than twice as long as that needed to test a sample in the cell-based assay. Other advantages of the cell-based method include the use of a 96-well format, which facilitates the analysis of several concentrations of rhTSH within one assay plate, and the use of a non-radioactive endpoint.


Asunto(s)
Bioensayo/métodos , Tirotropina/análisis , Tirotropina/farmacología , Animales , Células CHO , Bovinos , Cricetinae , Genes Reporteros , Humanos , Técnicas In Vitro , Luciferasas/genética , Radioinmunoensayo/métodos , Receptores de Tirotropina/genética , Proteínas Recombinantes/análisis , Proteínas Recombinantes/farmacología , Glándula Tiroides/efectos de los fármacos , Transfección
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