Measurement of Sialic Acid Content Is Insufficient to Assess Bioactivity of Recombinant Human Erythropoietin

Abstract

To achieve quality control of therapeutic proteins, it is important to compare their biological potency with their clinical effects.1) Therefore, an animal-based assay is preferred for measuring their bioactivity. However, the use of laboratory animals poses ethical concerns, is time consuming and requires skilled staff and sophisticated experimental facilities. Furthermore, in vivo bioassays have low precision and accuracy, and hence, there is a need for an alternative assay method to estimate in vivo bioactivity.To date, attempts have been made to replace traditional bioassays with physicochemical techniques for potency determination of insulin and growth hormone because it is possible to design and validate manufacturing processes in the production of properly folded materials.2,3) Potency of these pharmaceutical preparations can be calculated using the content assay with HPLC, which quantifies the peak area of a product against that of a reference standard of known potency. The results of the HPLC assay have been shown to correlate well with those of the bioassay.2,3) In addition, the HPLC assay is rapid, precise and less labour intensive than the bioassay. However, for some glycoproteins, alternative assay methods using a quantitative charge-based separation technique, such as isoelectric focusing (IEF) and capillary zone electrophoresis (CZE), have been shown to be feasible for estimating in vivo bioactivity 4,5) because negatively charged sialic acid residues are critical for in vivo bioactivity. For example, Mulders et al. 4) reported that quantitative assessment of the isoform distribution by IEF may provide a method for predicting in vivo biological potency of preparations containing recombinant human follicle-stimulating hormone (rhFSH). Close correlation between results of experimental in vivo bioactivities and IEF-predicted in vivo bioactivities suggest that the only factor affecting prediction of biological potency is the result of quantitative isoform distribution in rhFSH.Erythropoietin (EPO) is a glycoprotein hormone that regulates the red blood cell level by stimulating maturation of erythroid precursor cells. 6) Recombinant human EPO (rhEPO) produced in Chinese hamster ovary (CHO) cells is extensively used for serve anaemia therapy.7) EPO comprises a 165-amino acid protein with 40% of its molecular weight accounted for by carbohydrates.8,9) EPO contains three Nglycans located on Asn residues at positions 24, 38, and 83 and one mucin-type O-glycan located on the Ser-126 residue. The N-linked carbohydrates comprise bi-, tri-and tetraantennary oligosaccharides, which typically terminate with a negatively charged sialic acid residue. 10,11) Because of the variable number of sialic acid residues, the product of EPO usually exists as a mixture of isoforms with different isoelectric points. Several studies have reported that sialic acid residues are critical for in vivo bioactivity of EPO.12,13) Isoforms having a higher sialic acid content are seen to display higher in vivo bioactivity, longer seru...

Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

“…The glycoform distributions borne on each site cause great heterogeneities on the mature protein, whereas the absence of glycosylation greatly decreases the stability of the intermediate species, and changes the folding kinetics of erythropoietin[10]. The N -Glycan moieties, in particular the sialic acid residues that are generally attached at the non-reducing end of the glycans, have a significant impact on the in vivo bioactivity of rhEPO [11,12]. …”

Aggregate Forms of Recombinant Human Erythropoietin With Different Charge Profile Substantially Impact Biological Activities

“…17,33 On the other hand, some studies proposed that N-glycans with sialic acid extensions are not the main factors in of HuEPO biological activity. 23 Here, we studied and compared charge profiles that raising from the numbers of relative sialic acid content, protein structure, and particle size or aggregation on HuEPO biological activity based on the calculation of select sample I-number values.…”

“…20,21 Two other studies, Dordal et al and Delorme et al, revealed the importance of the glycosylation for solubility and stability of EPO. 16,22 Yanagihara et al, 23 however, reported that HuEPO activity might depend on other parameters in addition to I-Number or glycan moieties. Results from this work suggested that rHuEPOs possess comparable biological activities and similar secondary structure.…”

Evaluation of an In Vitro Cell Culture Assay for the Potency Assessment of Recombinant Human Erythropoietin

“…The hormone consists of a 165 amino acids polypeptide chain, heavily glycosylated at three N-linked and one O-linked glycosylation sites, with a molecular mass of 30.4kDa. About 40% of the fully glycosylated EPO molecule consists of carbohydrates, which play an important role in determining its biological activity (3,4).…”