The Relationship Between The Glycosylation Content And The Biological Activity Of Rhuepo

Recombinant human Erythropoietin is a glycoprotein hormone, which consists of 165amino acids with three N-glycosylation sites and one O-glycosylation site, and whichapproximately 40% by weight is sugar content. It is well known that the biological activityof rHuEPO is not only decided by the amino acids sequences but also in relation to theglycosylation content of rHuEPO, which is especially impacted by the sialic acid contendof sugar base. So the carbohydrate moiety is the linchpin of the rHuEPO quality. In theprocess of manufacturing, there are many aspects that can induce the difference ofcarbohydrate content of rHuEPO such as cell strains and manufacture technology. In orderto control the product quality, we must utility the biological activity as the decidingstandard. However, the disadvantage of the method is that prolonging the period of assayand the instability between experiment materials.For the purpose to forecast the product quality quickly and exactly it is importance toexplore the relationship between the experiment result in vitro and the biological activity.The six batches of rHuEPO bulk solution with different manufacture technology were usedto explore the relationship between the result of the following experiments: the first is tomeasure the protein content by lowery method; the second is to analyze the siatic acidcontent by resorcinol color assay; the third is to detect the biological activity in vitrothrough the enzyme-linked immunosorbent assay; the fourth is to assay pI through themethod of the isoelectric focusing electrophoresis; the next is reticulacyte counting method usedfor the detection of biological activity in vivo of rHuEPO; the last is the assay of capillaryzone electrophoresis to quantity the isoform content of rHuEPO. Analyze all the experimentdata to research the relationship between them by the SPSS 10.0 statistical analysissoftware. The result indicated that there were positive correlation between the sialic acidcontent and the biological activity in vivo and no correlation between the biological activityin vivo and the one in vivo. The correlation also exist between the pI and the biologicalactivity in vivo. In the study, Capillary zone electrophoresis allows the separation andquantitation of eight bands containing glycoforms of recombinant erythropoietin. The studysuggested that the proportion of the eight glycoforms of recombinant erythropoietin had the correlation with the ratio of the biological activity in vivo to the sialic acid content and theone of the biological activity in vitro to the sialic acid content. According the aboveanalyses result, the linear regression mathematics model were established among the majorimpact factors of glycoforms and the ration of the biological activity in vivo and in vitro tothe sialic acid content. The biological activity in vivo and in vitro could be predicted by themodels.