Two forms of erythropoietin, EPO-bi and EPO-tetra, with different biological activities were isolated from the culture medium of a recombinant Chinese hamster ovary cell line, B8-300, into which the human erythropoietin gene had been introduced. EPO-bi, an unusual form, showed only one-seventh the in vivo activity and 3 times higher in vitro activity of the previously described recombinant human EPO (standard EPO). In contrast, EPO-tetra showed both in vivo and in vitro activities comparable to those of the standard EPO. EPO-bi, EPO-tetra, and the standard EPO had the same amino acid composition and immunoreactivity. However, structural analyses of their N-linked sugar chains revealed that EPO-bi contains the biantennary complex type as the major sugar chain, while EPO-tetra and the standard EPO contain the tetraantennary complex type as the major sugar chain. From examination of various preparations of recombinant human EPO, we found a positive correlation between the in vivo activity of EPO and the ratio of tetraantennary to biantennary oligosaccharides. These results suggest that higher branching of the N-linked sugar chains is essential for effective expression of in vivo biological activity of EPO.Human erythropoietin (EPO) is a glycoprotein hormone that plays a major role in regulating the level of circulating erythrocytes (1) by stimulating the maturation of late erythroid progenitor cells into proerythroblasts (2). In the normal human adult, EPO is produced in the kidney (3). EPO was first purified in a small amount from urine of aplastic anemia patients (4). Several recombinant human EPOs produced in mammalian (5-9) and nonmammalian cells (10) have recently become available, but their biological activities differ from cell to cell. Since the structures of their polypeptide moieties are the same, such variation in activity was suspected as being due to the differences in their carbohydrate moieties. The sugar chain structures of urinary human EPO and recombinant human EPO produced in Chinese hamster ovary (CHO) cells were determined independently by us (11) and by Sasaki et al. (12). Both urinary and recombinant human EPOs contain -40% carbohydrate in the form of three N-linked and one O-linked oligosaccharide chain. Important roles of the carbohydrate moiety in the solubility (13, 14), biosynthesis (14), and biological activity of EPO (9, 15) have been reported. Desialylation of EPO caused complete loss of its hormonal activity in vivo (15, 16) as the asialo-EPO was trapped in the liver (17) by the hepatic asialoglycoprotein binding protein (18) and was rapidly cleared from the circulation. Galactose oxidase treatment of asialo-EPO restored part of the biological activity (16). These results suggested that EPO possesses full biological activity only when it is sufficiently sialylated to avoid clearance by the hepatic asialoglycoprotein binding protein. During the course of study of the productivity of EPO in several recombinant CHO cell lines, we found a unique cell line, B8-300, which produced ...

Physicochemical properties of recombinant human erythropoietin were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human urine when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Sedimentation equilibrium experiments showed the recombinant erythropoietin preparation to be essentially a single macromolecular component with a molecular weight of 30,400 and a carbohydrate content of 39%. The Stokes radius of recombinant erythropoietin was estimated to be 32 A from gel filtration, much larger than the 20-A radius calculated for a sphere of the observed molecular weight. This difference may be ascribed to the extensive glycosylation. The fluorescence and phosphorescence spectra showed that the luminescent tryptophan(s) is (are) solvent-exposed and can be quenched by I- and acrylamide but not by Cs+. On acid titration, the recombinant erythropoietin showed a conformational transition with a midpoint of pH 4.1. This suggests that the net charges on the protein moiety rather than on the whole molecule play a role in protein structure stability.

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High-performance capillary electrophoresis (HPCE) has been employed to characterize the peptide map of recombinant human erythropoietin (rHuEPO) expressed from Chinese hamster ovary (CHO) cells. The methodology employs an ion pairing agent, 100 mM heptanesulfonic acid in 40 mM sodium phosphate buffer, pH 2.5, to increase peptide resolution, to decrease analyte wall interactions, and to evaluate glycopeptide microheterogeneity. The total tryptic map is segregated into two regions, nonglycosylated and glycosylated peptides. Reproducibility of the peptide map is excellent; the map results in baseline separation of 16 tryptic peptides and one doublet peak composed of two peptides (resolution 0.22). The map furthermore allows for the evaluation of the microheterogeneity associated with the three rHuEPO glycopeptides. At least 12 glycopeptide forms were separated in the initial peptide map. Peptides were identified by Edman sequencing, and the glycopeptides were further subjected to Dionex anion-exchange chromatography. To simplify the level of complexity associated with the glycopeptides, much of the characterization employed asialoglycopeptides and employed several endoproteolytic diagnosis. The relative percent distribution for each purified asialoglycopeptide was calculated to define the level of complexity and to tentatively assign a known structure to the HPCE peak. The level of structural complexity of the asialoglycopeptides appears to increase from the simplest O-linked form to the more complex N83, N38, and N24 glycosylation positions, respectively. HPCE evaluation of glycopeptide microheterogeneity appears to be simpler, faster, and just as sensitive as other more frequently employed methods for glycopeptide characterizations.