Detection of isoelectric profiles of erythropoietin in urine: differentiation of natural and administered recombinant hormones

Erythropoietin (EPO) is normally present in urine at a low concentration (about 1IU/L, i.e., about 10ng/L) for a total protein concentration of at least 50mg/L. A method to study the isoelectric profile of this hormone from 20-ml urine aliquots without previous purification was developed. This method involves isoelectric focusing of the retentate from ultrafiltered urine. Both the ultrafiltration and the isoelectric focusing required precautionary measures to prevent EPO degradation by the proteases that are present in urine. Because classical immunoblotting gave rise to an unspecific detection of various urinary proteins in the focused retentate, it was essential to use the "double-blotting" process developed to solve this problem. Sufficient sensitivity was achieved using amplified chemiluminiscent detection after the blotting membrane was treated with dithiotreitol. The patterns that were revealed from various urinary samples proved to be highly heterogeneous as they were composed of more than 10 isoforms in a pI range of 3.7-4.7. Clear transformation of the patterns was observed in the case of treatment by the recombinant hormone, suggesting that this method can be regarded an efficient tool for indicating recombinant EPO misuse in sports. It may also open new investigations in the field of physiologic or pathologic exploration.     

Rapid detection of erythropoiesis-stimulating agents in urine and serum

A rapid and easy-to-use test kit, EPO WGA MAIIA, which can be used for distinguishing various endogenous human erythropoietins (hEPOs) and several recombinant hEPO and EPO analogues, has been evaluated. The test is based on chromatographic separation of the glycosylated isoforms of EPO using wheat germ agglutinin (WGA) and a sensitive immunoassay using anti-EPO carbon black nanostrings and image scanning for quantification. All of the reactions take place along the porous layer of a lateral flow microcolumn containing WGA and anti-EPO zones. The presence of molecules resembling hEPOs, such as Mircera, was detected by the aberrant affinity interaction with the antibody zone on the strip. It was possible to distinguish nine recombinant hEPOs expressed in hamster and human cell lines, as well as Aranesp and Mircera, from endogenous urine hEPO. The required amount of EPO in the samples, a few picograms, is very low compared with other methods for EPO isoform identification. This EPO isoform determination method opens the possibility to monitor recombinant EPO therapy for clinical research and seems to be a valuable candidate to the arsenal of EPO doping control tests.     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

Effect of constant light on prolactin and corticosterone rhythms evaluated using a noninvasive urine sampling protocol in the rat.

Circadian prolactin and corticosterone rhythms are usually investigated in the rat by analysis of plasma hormone profiles. In order to develop a nonstressful methodology for long-term studies, we validated prolactin and corticosterone radioimmunoassays in rat urine samples. Among the criteria of validation, prolactin was identified in urine by Western blot whereas both prolactin and corticosterone levels were undetectable in the urine of hypophysectomized rats. The determination of prolactin and corticosterone levels on serial urine samples showed daily variations in male rats entrained by the light-dark cycle. The acrophases of the 24-hour prolactin and corticosterone profiles were located at 03:26 h and 23:32 h respectively, a delay of 3-4 hours compared with the values of the 24-hour plasma profiles reported in the literature. Corticosterone and prolactin rhythms were abolished or dramatically delayed after 3 weeks of constant illumination. As expected, constant light suppressed the rhythm of 6-sulfatoxymelatonin, the major hepatic metabolite of melatonin. The noninvasive and nonstressful methodology we developed could be of interest for studying the regulation of hormone rhythms and their mutual endocrine interactions in physiological conditions, especially their evolution in the aging process.     

Efficient purification and preconcentration of erythropoietin in human urine by reusable immunoaffinity column

In this paper, an efficient method is proposed for purification and preconcentration of erythropoietin (EPO) in human urine samples. The EPO-specific immunoaffinity column (IAC) was generated by covalent immobilization of anti-EPO polyclonal antibodies on Sepharose 4B support. The EPO-binding capacity of the IAC was found to be about 2.0 microg (6.6IU) per 1.5 mL of gel and the activity recoveries of EPO at low concentrations of 7.8, 10 and 120 m IU/mL by the IAC were between 78 and 86%. Substantial cleanup effect was observed when the IAC was applied to human urine samples.     

Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin. Identification of des-arginine 166 erythropoietin

Recombinant human erythropoietin (rhEPO) has been purified to apparent homogeneity from a Chinese hamster ovary cell line expressing a cDNA clone of the human gene. NH2-terminal sequencing of the recombinant hormone indicates that the 27-residue leader peptide is correctly and consistently cleaved during secretion of the recombinant protein into conditioned medium, yielding the mature NH2 terminus (Ala-Pro-Pro-Arg...). Analysis of the COOH terminus of rhEPO by peptide mapping and fast atom bombardment mass spectrometry (FABMS) demonstrates that the arginyl residue predicted to be at the COOH terminus (based on confirmation of both genomic and cDNA sequences) is completely missing from the purified protein. The truncated form of the recombinant hormone, designated des-Arg166 rhEPO, displays an in vivo specific activity of greater than 200,000 units/mg protein. Structural characterization of natural human urinary EPO (uEPO) by peptide mapping and FABMS reveals that the urinary hormone is also missing the COOH-terminal Arg166 amino acid residue, a modification that remained undetected until now. There is no evidence of further proteolytic processing at the COOH terminus beyond specific removal of the Arg166 amino acid residue in either rhEPO or uEPO. On the basis of the FABMS data, we propose that the physiologically active form of the hormone circulating in plasma and interacting with target cells in vivo is des-Arg166 EPO.     

Properties of a cleaved two-chain form of recombinant human growth hormone

Escherichia coli cells transformed with plasmids engineered for the expression of recombinant human growth hormone as a secreted product also produced a proteolytically cleaved form of rhGH. This variant is isolated at a high resolution anion exchange chromatography stage during the manufacturing process. The higher isoelectric point of this form is demonstrated by isoelectric focusing and chromatofocusing and the two-chain nature by tryptic mapping, N- and C-terminal sequence analyses, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These data indicate that the single site of cleavage is between Thr-142 and Tyr-143, in contrast to the two-chain variant isolated from human pituitary glands, which has a clip after residue Phe-139. The recombinant two-chain form was further characterized by reversed-phase high performance liquid chromatography at both acidic and basic pHs. The assay utilizing bicarbonate-containing mobile phases was determined to be the most efficient and sensitive method. The bioactivity of this two-chain form was measured by the in vivo rat weight gain assay and by the in vitro Nb2 cell bioassay. Its immunological similarity to intact one-chain rhGH was demonstrated with an enzyme-linked immunosorbent assay.     

Human Ubiquitin C Promoter Based Expression of Erythropoietin in CHO K1 Cell Lines: A Simple Transfectants Screening Approach

Erythropoietin (EPO), a glycoprotein hormone that regulates the production of erythrocytes in the human body, is of clinical importance in the treatment of anemia. Low expression levels of this recombinant hormone and time-consuming screening methods have made its commercial production expensive. Cloning of human EPO gene in a shuttle vector pUB6/V5-HisB driven by human ubiquitin C promoter and its transfection in CHO K1 cell lines by electroporation resulted in a moderate level of EPO expression. The limiting-dilution screening method required several months to obtain high expression stable transfectants but needed only short duration for selection in contrast to the present screening strategy. The supernatants of stably transfected cells were found to be biologically active by in vitro erythroid cluster forming activity.     

Characterization of intact glycopeptides reveals the impact of culture media on site-specific glycosylation of EPO-Fc fusion protein generated by CHO-GS cells

With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N- and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N- and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc N-glycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi- and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1–4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N- and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest.     

人促红细胞生成素基因在家蚕体中的高效表达

人促红细胞生成素(EPO)是一种调控红系干细胞增殖、分化和成熟的糖蛋白激素。将合成的EPO cDNA插入杆状病毒转移载体pBlueBacⅢ,使其置于Ph基因强启动子控制之下,获得了转移载体pBlueBacEPO。将pBlueBacEPO DNA与野生型BmNPV DNA共转染BmN细胞,经空斑纯化,获插入EPO cDNA的重组病毒rBmNPVEPO。经Sonthern杂交和PCR扩增鉴定证明人EPO基因已正确组建于BmNPV的预定位置。将重组病毒rBmNPVEPO穿刺接种5龄幼虫和蛹,收集感染第3～5d的幼虫血淋巴和3～6.5d蛹血淋巴。用ELISA检测幼虫血淋巴中EPO表达量高达62800u/mL,蛹血淋巴中表达量达74000u/mL。Western blot结果显示幼虫血淋巴和蛹血淋巴均有一条明显的免疫杂交带,分子量均约为26kD。用TF1细胞对幼虫表达产物进行了生物活性测定,每毫升血淋巴中EPO活性约为63000u。     

Effect of culture temperature on erythropoietin production and glycosylation in a perfusion culture of recombinant CHO cells

To investigate the effect of culture temperature on erythropoietin (EPO) production and glycosylation in recombinant Chinese hamster ovary (CHO) cells, we cultivated CHO cells using a perfusion bioreactor. Cells were cultivated at 37 degrees C until viable cell concentration reached 1 x 10(7) cells/mL, and then culture temperature was shifted to 25 degrees C, 28 degrees C, 30 degrees C, 32 degrees C, 37 degrees C (control), respectively. Lowering culture temperature suppressed cell growth but was beneficial to maintain high cell viability for a longer period. In a control culture at 37 degrees C, cell viability gradually decreased and fell below 80% on day 18 while it remained over 90% throughout the culture at low culture temperature. The cumulative EPO production and specific EPO productivity, q(EPO), increased at low culture temperature and were the highest at 32 degrees C and 30 degrees C, respectively. Interestingly, the cumulative EPO production at culture temperature below 32 degrees C was not as high as the cumulative EPO production at 32 degrees C although the q(EPO) at culture temperature below 32 degrees C was comparable or even higher than the q(EPO) at 32 degrees C. This implies that the beneficial effect of lowering culture temperature below 32 degrees C on q(EPO) is outweighed by its detrimental effect on the integral of viable cells. The glycosylation of EPO was evaluated by isoelectric focusing, normal phase HPLC and anion exchange chromatography analyses. The quality of EPO at 32 degrees C in regard to acidic isoforms, antennary structures and sialylated N-linked glycans was comparable to that at 37 degrees C. However, at culture temperatures below 32 degrees C, the proportions of acidic isoforms, tetra-antennary structures and tetra-sialylated N-linked glycans were further reduced, suggesting that lowering culture temperature below 32 degrees C negatively affect the quality of EPO. Thus, taken together, cell culture at 32 degrees C turned out to be the most satisfactory since it showed the highest cumulative EPO production, and moreover, EPO quality at 32 degrees C was not deteriorated as obtained at 37 degrees C.     

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Human recombinant erythropoietin (rHuEPO) was produced from Chinese hamster ovary (CHO) cells transfected with the human EPO gene. The cells were grown in batch cultures in controlled bioreactors in which the set-points for dissolved oxygen varied between 3% and 200%. The cell-specific growth rate and final cell yield was significantly lower under hyperoxic conditions (200% DO). However, there was no significant difference in growth rates at other oxygen levels compared to control cultures run under a normoxic condition (50% DO). The specific productivity of EPO was significantly lower at a DO set-point of 3% and 200% but maintained a consistently high value between 10% to 100% DO. The EPO produced under all conditions as analyzed by two-dimensional electrophoresis showed a molecular weight range of 33 to 37 kDa and a low isoelectric point range of 3.5 to 5.0. This corresponds to a highly glycosylated and sialylated protein with a profile showing at least seven distinct isoforms. The glycan pattern of isolated samples of EPO was analyzed by weak anion exchange (WAX) HPLC and by normal-phase HPLC incorporating sequential digestion with exoglycosidase arrays. Assigned structures were confirmed by mass spectrometry (MALDI-MS). The most prominent glycan structures were core fucosylated tetranntenary with variable sialylation. However, significant biantennary, triantennary, and non-fucosylated glycans were also identified. Detailed analysis of these glycan structures produced under variable dissolved oxygen levels did not show consistently significant variations except for the ratio of fucosylated to non-fucosylated isoforms. Maximum core fucosylation (80%) was observed at 50% and 100% DO, whereas higher or lower DO levels resulted in reduced fucosylation. This observation of lower fucosylation at high or low DO levels is consistent with previous data reported for glycoprotein production in insect cells.     

Physicochemical and biological comparison of recombinant human erythropoietin with human urinary erythropoietin

Physicochemical and biological properties of recombinant human erythropoietin (rhEPO) were compared with human urinary erythropoietin (uEPO). uEPO and rhEPO were purified to apparent homogeneity from the urine of patients with aplastic anemia and from the conditioned medium of Chinese hamster ovary (CHO) cells transfected with a cDNA clone for human EPO, respectively. The microheterogeneous nature of both factors, observed on isoelectric focusing, is derived from the difference of the number of terminal sialic acid residues bound to the carbohydrate chains of the EPO molecule. The primary structure of rhEPO, consisting of 165 amino acid residues, was determined, and the C-terminal arginine predicted from the cDNA sequence was confirmed to be missing, as described previously (Recny et al. (1987) J. Biol. Chem. 262, 17156). Three N-glycosylation and one O-glycosylation sites of both factors were determined as Asn24, Asn38, and Asn83 and Ser126, respectively. Two disulfide linkages are located between Cys7 and Cys161, and between Cys29 and Cys33, in both EPOs. Hematogenic potencies of rhEPO and uEPO compared in normal and in partially nephrectomized rats were approximately the same. Both factors also stimulated the colony formation of CFU-E, BFU-E, and CFU-Meg in a dose-dependent manner. From these results, it is concluded that rhEPO produced in CHO cells transfected with cDNA clone for human EPO is indistinguishable from uEPO physicochemically and biologically, and is valuable for further research and for clinical use.     

Two-dimensional gel electrophoresis for controlling and comparing culture supernatants of mammalian cell culture productions systems

A recombinant Chinese hamster ovary cell line, producing human erythropoietin, was cultivated in a continuous mode in a stirred tank reactor applying different dilution rates. In order to monitor the stability of this expression system, product and non-product proteins of the cell culture supernatant were analyzed by two-dimensional electrophoresis. The consistency of the isoforms of the recombinant product was determined by western blot combined with specific staining.The same cell line was propagated in a high cell density cultivation system based on macro-cell-aggregates. The patterns of secreted proteins of the cell line cultivated in the different systems were compared in order to detect modifications in protein expression of the product and of non product proteins relevant for cell culture supernatant.Hardly any alterations in two-dimensional pattern were detectable. The isoforms of erythropoietin, as well as the overall pattern of secreted proteins, detectable with the two-dimensional electrophoresis method were remarkably stable under different cultivation conditions.Abbreviations 2-DE two-dimensional electrophoresis - rCHO recombinant Chinese hamster ovary cell - EPO erythropoietin - FBR fluidized bed reactor - IEF isoelectric focusing - IPG immobilized pH gradient - pI isoelectric point - SDS sodium dodecylsulfate - STR stirred tank reactor     

Impact of host cell line choice on glycan profile

Protein glycosylation is post-translational modification (PTM) which is important for pharmacokinetics and immunogenicity of recombinant glycoprotein therapeutics. As a result of variations in monosaccharide composition, glycosidic linkages and glycan branching, glycosylation introduces considerable complexity and heterogeneity to therapeutics. The host cell line used to produce the glycoprotein has a strong influence on the glycosylation because different host systems may express varying repertoire of glycosylation enzymes and transporters that contributes to specificity and heterogeneity in glycosylation profiles. In this review, we discuss the types of host cell lines currently used for recombinant therapeutic production, their glycosylation potential and the resultant impact on glycoprotein properties. In addition, we compare the reported glycosylation profiles of four recombinant glycoproteins: immunoglobulin G (IgG), coagulation factor VII (FVII), erythropoietin (EPO) and alpha-1 antitrypsin (A1AT) produced in different mammalian cells to establish the influence of mammalian host cell lines on glycosylation.     

Effects of ammonia and glucosamine on the heterogeneity of erythropoietin glycoforms

Recombinant human erythropoietin (EPO) is a glycoprotein produced as a therapeutic agent from mammalian cell cultures for the treatment of anemia associated with severe kidney damage. The EPO structure has a high glycan content which is essential for bioactivity but shows considerable molecular heterogeneity. The cell culture conditions that affect the heterogeneity of the glycoforms of EPO are not well understood. However, the accumulation of ammonia in culture is one factor that has been associated with an enhanced heterogeneity of glycoforms. In this report we investigate the metabolic perturbations associated with ammonia and glucosamine that may give rise to an altered pattern of EPO glycosylation. Recombinant human erythropoietin was synthesized in serum-free cultures of transfected Chinese hamster ovary (CHO) cells. The molecular heterogeneity of erythropoietin was increased by supplementation of cultures with either ammonia or glucosamine. The enhanced molecular heterogeneity was shown to be due to variable glycosylation that resulted in EPO with an enhanced molecular weight and isoelectric point range. Enzymatic removal of the glycan moiety of EPO in all cases resulted in a single molecular form with a molecular weight of 18 000, which corresponded to non-glycosylated EPO. The variable glycosylation was consistent with reduced sialylation and antennarity of the carbohydrate structures present on the three N-linked sites of EPO. In the presence of ammonia (>30 mM) the proportion of tetrasialylated and tetraantennary glycan structures were reduced by 73% and 57%, respectively, as determined by HPLC analysis. Such changes were also observed, although to a lesser extent (41% and 37%), by an increase in the glucosamine concentration (>10 mM) in the medium. The enhanced heterogeneity of the glycan structures coincided with a significant increase in the intracellular UDP-N-acetylhexosamine (UDP-GNAc) pool. The measured UDP-GNAc level was up to 2 orders of magnitude higher in the presence of either glucosamine or ammonia. However, the changes in the glycosylation profiles induced by either glucosamine or ammonia were significantly different even at the same intracellular UDP-GNAc concentration. This suggests that the enhanced EPO heterogeneity could not be mediated solely by the increased UDP-GNAc level. Glucosamine (but not ammonia) was shown to cause significant inhibition of glucose transport into the cells, which could induce a different pattern of primary metabolism.     

Structures and functional roles of the sugar chains of human erythropoietins.

Erythropoietin (EPO) is a haemopoietic hormone specific to cells of erythroid lineage. EPO has recently become available for the treatment of anaemia as the first human recombinant biomedicine produced in heterologous mammalian cells. Human EPO is characterized by its large carbohydrate chains, which occupy close to 40% of its total mass. These sugar moieties were thought to be important for the biological activity of EPO, but detailed studies were not performed until the structures were elucidated. The variety of roles for the sugar chains were then immediately found once the structures were known. EPO is an excellent model for investigating the roles of sugar chains on glycoproteins, since its gene and its multiple glycoforms are available, as well as sensitive bioassays for testing. In this review, we will first summarize the known sugar chain structures of EPO from different host cells, and then discuss the host-cell dependent and peptide structure-dependent glycosylation of glycoproteins. We will then address how one investigates the roles of sugar chains of glycoproteins, show several examples of such investigations, and discuss the functional roles of HuEPO's sugar chains in its biosynthesis and secretion, its in vitro and in vivo biological activities, and its half-life in blood circulation.     

Detection of bladder cancer using proteomic profiling of urine sediments

We used protein expression profiles to develop a classification rule for the detection and prognostic assessment of bladder cancer in voided urine samples. Using the Ciphergen PBS II ProteinChip Reader, we analyzed the protein profiles of 18 pairs of samples of bladder tumor and adjacent urothelium tissue, a training set of 85 voided urine samples (32 controls and 53 bladder cancer), and a blinded testing set of 68 voided urine samples (33 controls and 35 bladder cancer). Using t-tests, we identified 473 peaks showing significant differential expression across different categories of paired bladder tumor and adjacent urothelial samples compared to normal urothelium. Then the intensities of those 473 peaks were examined in a training set of voided urine samples. Using this approach, we identified 41 protein peaks that were differentially expressed in both sets of samples. The expression pattern of the 41 protein peaks was used to classify the voided urine samples as malignant or benign. This approach yielded a sensitivity and specificity of 59% and 90%, respectively, on the training set and 80% and 100%, respectively, on the testing set. The proteomic classification rule performed with similar accuracy in low- and high-grade bladder carcinomas. In addition, we used hierarchical clustering with all 473 protein peaks on 65 benign voided urine samples, 88 samples from patients with clinically evident bladder cancer, and 127 samples from patients with a history of bladder cancer to classify the samples into Cluster A or B. The tumors in Cluster B were characterized by clinically aggressive behavior with significantly shorter metastasis-free and disease-specific survival.     

Optimization of parameters affecting on CHO cell culture producing recombinant erythropoietin

Abstract

Several factors may affect erythropoietin (EPO) sugar structures including designing cell culture procedure, pH, concentration of additives, dissolved oxygen, and other physicochemical parameters. In this study, we investigated the influence of changes in effective parameters and compounds on the growth rate of Chinese hamster ovary cell (CHO) cells producing recombinant EPO. Cell culture was performed at different temperature, buffering conditions, and varied concentrations of additives such as pyruvic acid, insulin, GlutaMAX, and sodium butyrate. Results indicated that the optimal temperature and pH were 37?°C and 7.2, respectively. Also, optimal concentrations for pyruvic acid, butyrate, glutamate, and insulin were obtained to be 20?mM, 1?mM, 2?mM, and 40?μg/mL, respectively. Then, cell culture was performed in microcarrier-coated spinner flasks under the optimized condition. The results showed recombinant human EPO (rhEPO) production with adequate purity. Optimization of physicochemical conditions and culture media are important factors to improve the quantity and quality of protein products. This study showed that cell growth and recombinant EPO protein production significantly increased under the optimized conditions. The results of this research can also be used in scale-up to increase the efficiency of EPO production.

Abbreviations: EPO: erythropoietin; CHO cell: Chinese hamster ovary cell; rhEPO: recombinant human EPO; DMEM: modified eagle’s medium; FBS: fetal bovine serum; SDS-PAGE: sodium dodecyl sulfate–polyacrylamide gel electrophoresis; IGF-1: insulin-like growth factor 1