Production Of Recombinant Human Erythropoietin Research Articles

Recombinant Human Erythropoietin Production in Chinese Hamster Ovary Cells Is Enhanced by Supplementation of α-Helix Domain of 30Kc19 Protein

The enhancement of recombinant therapeutic protein production in mammalian cell culture has been regarded as an important issue in the biopharmaceutical industry. Previous studies have reported that the addition of the recombinant 30Kc19 protein, a silkworm-derived plasma protein with simultaneous cell-penetrating and mitochondrial enzyme-stabilizing properties, can enhance the recombinant protein expression in Chinese hamster ovary (CHO) cell culture. Here, we produced an α-helix N-terminal domain of 30Kc19, called (30Kc19α), and investigated its effects on the production of human erythropoietin (EPO), a widely used therapeutic protein for the treatment of anemia, in recombinant CHO cell culture. Similar to the full-length 30Kc19, 30Kc19α was able to be mass-produced in a form of recombinant protein through an Escherichia coli expression system and delivered into EPO-producing CHO (EPO–CHO) cells. Supplementing the medium of EPO–CHO cell culture with 30Kc19α increased the intracellular NADPH/NADP+ ratio related to the flux of metabolic reducing power for protein biosynthesis, subsequently enhancing EPO production in serum-free culture. 30Kc19α is considered to have certain advantages in the downstream purification process of therapeutic protein production when it is used as a medium supplement due to its small size and low isoelectric point compared to the full-length 30Kc19. These results suggest that 30Kc19α has potential use for manufacturing biopharmaceutical proteins.

High-throughput engineering of initial coding regions for maximized production of recombinant proteins

We present a method to rapidly determine the optimal initial sequences for recombinant protein production. This method relies on the competitive translation of two genes in a reaction mixture for cell-free protein synthesis. Genes from a library with randomized +2/+3 codons were individually co-expressed with superfolder green fluorescent protein (sfGFP) in this reaction mixture. As a result of competitive translation, the intensity of sfGFP fluorescence was inversely proportional to the relative translational efficiency of the co-expressed gene. Using this simple method, we identified the +2/+3 codons that markedly enhanced production of recombinant human erythropoietin compared with its wild-type codons. These +2/+3 codons also effectively enhanced protein production in Escherichia coli, indicating that this method is a useful tool to design optimal gene constructs for industrial production of recombinant proteins.

Erythropoietin action in stress response, tissue maintenance and metabolism.

Erythropoietin (EPO) regulation of red blood cell production and its induction at reduced oxygen tension provides for the important erythropoietic response to ischemic stress. The cloning and production of recombinant human EPO has led to its clinical use in patients with anemia for two and half decades and has facilitated studies of EPO action. Reports of animal and cell models of ischemic stress in vitro and injury suggest potential EPO benefit beyond red blood cell production including vascular endothelial response to increase nitric oxide production, which facilitates oxygen delivery to brain, heart and other non-hematopoietic tissues. This review discusses these and other reports of EPO action beyond red blood cell production, including EPO response affecting metabolism and obesity in animal models. Observations of EPO activity in cell and animal model systems, including mice with tissue specific deletion of EPO receptor (EpoR), suggest the potential for EPO response in metabolism and disease.

Highly sialylated recombinant human erythropoietin production in large‐scale perfusion bioreactor utilizing CHO‐gmt4 (JW152) with restored GnT I function

Therapeutic glycoprotein drugs require a high degree of sialylation of their N-glycans for a better circulatory half-life that results in greater efficacy. It has been demonstrated that Chinese hamster ovary (CHO) glycosylation mutants lacking N-acetylglucosaminyltransferase I (GnT I), when restored by introduction of a functional GnT I, produced highly sialylated erythropoietin (EPO). We have now further engineered one of such mutants, JW152, by inactivating the dihydrofolate reductase (DHFR) gene to allow for the amplification of the EPO gene with methotrexate (MTX). Several MTX-amplified clones maintained the ability to produce highly sialylated EPO and one was selected for culture in a perfusion bioreactor that is used in an existing industrial EPO-production bioprocess. Extensive characterization of the EPO produced was performed using total sialic quantification, HPAEC-PAD and MALDI-TOF MS analyses. Our results demonstrated that the EPO produced by the mutant line exhibits superior sialylation compared to the commercially used EPO-producing CHO clone cultured under the same conditions. Therefore, this mutant has the industrial potential for producing highly sialylated recombinant EPO and potentially other recombinant glycoprotein therapeutics.

Co‐substrate mannitol feeding strategy design in semi‐batch production of recombinant human erythropoietin production by Pichia pastoris

AbstractBACKGROUNDThe effects of alternative co‐substrate feeding strategies on recombinant human erythropoietin (rHuEPO) production by Pichia pastoris‐Mut+ strain were investigated.RESULTSFive different production strategies were designed and performed in the production phase of the bioprocesses with the use of either mannitol or sorbitol as the co‐substrate. The highest rHuEPO production was achieved as CrHuEPO= 0.65 g L−1 at t=9 h, with the cell concentration Cx=55 g L−1 in the production phase; wherein methanol was fed to the bioreactor with a predetermined dynamic feeding rate calculated for constant μM0=0.03 h−1; and three consecutive pulses of the co‐substrate mannitol were introduced at t=0, 6, and 12 h, so that CMan=50 g L−1. The overall cell and product yields on the substrates methanol and mannitol were found, respectively, as YX/St=0.22 g g−1 and YrHuEPO/St=3.74 mg g−1.CONCLUSIONSThe use of mannitol as the co‐substrate enhanced rHuEPO production and furthermore shortened the bioprocess time. The design of semi‐batch feeding strategy, based on the selection of the substrates and the co‐substrates, and their dynamic feeding into the bioreactor, is important to increase the production and productivity in r‐protein production by Mut+ strains of P. pastoris, and the developed strategy would be especially important for therapeutic glycoprotein productions by P. pastoris. © 2013 Society of Chemical Industry

Transient erythropoietin overexpression with cucumber mosaic virus suppressor 2b in Nicotiana benthamiana

Erythropoietin (EPO) is a glycoprotein hormone that regulates red blood cell formation in mammals. It also plays an important role in wound healing and the brain’s response to neuronal injury. Recombinant EPO has been produced in various hosts including Escherichia coli, insect cells, and yeast. Plant systems are also useful for the production of recombinant proteins. In this study, we used Nicotiana benthamiana for the production of recombinant human EPO (rhEPO) using an Agrobacterium-mediated transient expression system. We evaluated the effect of repeated agroinfiltration and Agrobacterium density on the rhEPO production. In addition, the rhEPO expression vector was coinfiltrated with a vector expressing cucumber mosaic virus suppressor 2b (CMV2b), which suppresses posttranscriptional gene silencing. We found that rhEPO expression was increased approximately 5.5-fold in N. benthamiana leaves coinfiltrated with CMV2b. In contrast, neither Agrobacterium density nor the number of infiltrations influenced rhEPO expression.

Optimization of CHO cell culture medium for production of recombinant human erythropoietin and interferon beta-1a

Optimization of CHO cell culture medium for production of recombinant human erythropoietin and interferon beta-1a

Enhanced recombinant human erythropoietin production by Pichia pastoris in methanol fed-batch/sorbitol batch fermentation through pH optimization

The effects of pH on recombinant human erythropoietin (rHuEPO) production by Pichia pastoris was investigated at pH = 4.0, 4.5, 5.0, 5.5 and 6.0, where sorbitol and methanol were supplied to the bioreactor in batch and fed-batch mode, respectively, in the recombinant protein production phase (PP). For the first three phases, glycerol batch (GB), glycerol fed-batch (GFB) and methanol transition (MT), the growth profiles were almost the same for pH ≥ 5.0; however, at pH < 5.0 the growth rate was lower. The highest cell concentration was obtained as 81.4 g L −1 where GB and GFB were operated at pH = 5.0 and MT and PP were operated at pH = 4.5. Sorbitol added at the beginning of PP, was totally consumed within 15 h, at pH ≤ 5.0; however, at pH > 5.0 sorbitol consumption rate decreased. The highest rHuEPO concentration was achieved at pH = 4.5 as 0.16 g L −1, where the overall cell and rHuEPO yield on substrate, were 0.5 g g −1 and 1.5 mg g −1. pH did not have a significant effect on extracellular protease activity. The highest specific AOX activity (110 U g −1 CDW) was attained at pH = 4.5 parallel to the highest rHuEPO production. At pH = 4.5, higher oxygen demand of the cells resulted in higher oxygen K La, 0.11 s −1, values.

Sports anaemia, regulation of erythropoietin biosynthesis during physical effort and EPO doping

Efficient transport of oxygen within the body is of particular importance for physical work performance, maximal aerobic power and resistance to fatigue, especially in endurance disciplines such as cycling, long...

Survival and proliferative roles of erythropoietin beyond the erythroid lineage

Since the isolation and purification of erythropoietin (EPO) in 1977, the essential role of EPO for mature red blood cell production has been well established. The cloning of the EPO gene and production of recombinant human EPO led to the widespread use of EPO in treating patients with anaemia. However, the biological activity of EPO is not restricted to regulation of erythropoiesis. EPO receptor (EPOR) expression is also found in endothelial, brain, cardiovascular and other tissues, although at levels considerably lower than that of erythroid progenitor cells. This review discusses the survival and proliferative activity of EPO that extends beyond erythroid progenitor cells. Loss of EpoR expression in mouse models provides evidence for the role of endogenous EPO signalling in nonhaematopoietic tissue during development or for tissue maintenance and/or repair. Determining the extent and distribution of receptor expression provides insights into the potential protective activity of EPO in brain, heart and other nonhaematopoietic tissues.

Use of Biodiesel Byproduct Crude Glycerol as the Carbon Source for Fermentation Processes by Recombinant Pichia pastoris

The aim of this study is to investigate the efficiency of using crude glycerol, the main byproduct of biodiesel industry, directly in the production media of Pichia pastoris fermentation processes. Different biodiesel synthesis conditions were examined, that is, transesterifications of canola, corn, soybean, and sunflower oils, with pure methanol in a molar ratio of 1:6 or 1:3, using 1% or 0.5% (w/v) NaOH as the catalyst. Among these, canola oil-derived glycerol served as the most favorable carbon source, and at a 1:6 molar ratio of canola oil:methanol and CNaOH = 1% (w/v), the highest recombinant human erythropoietin production (CrEPO = 31 mg L-1), cell concentration (CX = 10.5 g L-1), product yield on substrate (YrEPO/S = 1.48 mg g-1), and cell yield on substrate (YX/S = 0.57 g g-1) were obtained, having values approximately 1.4-fold higher than those attained with pure glycerol. Thus, this study demonstrated a potential use for crude glycerol, without any purification, in the production of additional v...

INTERNAL MEDICINE, RENAL ANAEMIA, AND ERYTHROPOIESIS-STIMULATING AGENTS (ESAS)

The connection between hypoxaemia and red blood cell (RBC) production was already known at the end of the 19th century. In 1883, Miescher (1) observed an increase in his own haemoglobin (Hb) concentration and red cell count when entering a sanatorium in Arosa (Swiss Alps). He realized that this increase occurred in response to reduced oxygen availability. The fascinating history of the development of erythropoietin (EPO) has been summarized by Denker (2) and more recently by Nanagaku and Eckardt (3). Erslev (4) elegantly demonstrated that plasma from anaemic rabbits contained a factor capable of stimulating RBS production and predicted its therapeutic potential. The human EPO gene was cloned in 1983, and this enabled the expression of the mature glycoprotein hormone from Chinese hamster ovary cells and the mass production of recombinant human erythropoietin (rhEPO – called epoietin in the literature) to be available for clinical use (5;6). The fi rst successful clinical treatments with EPO in haemodialysis (HD) patients were reported in 1986-1987 (7;8). As might be expected with a genetic copy of the endogenous protein, EPO is highly effective in stimulating erythropoiesis, particularly in chronic renal failure, and has very low immunogenicity (see below). Over the last years, novel erythropoiesis-stimulating agents (ESAs) have been developed (see below).

Production of recombinant human erythropoietin from Pichia pastoris and its structural analysis

To design and investigate a recombinant expression system producing a therapeutically important glycoprotein, human erythropoietin (rHuEPO), by Pichia pastoris. EPO cDNA was cloned into pPICZalphaA for expression under control of AOX1 promoter and fused, on the amino-terminal end, with a polyhistidine tag for rapid purification. A target site for factor Xa protease was also introduced, such that cleavage in vitro produced a mature form of rHuEPO having the native N- and C-termini. RHuEPO was characterized as to the extent and nature of N-linked glycosylation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and western blotting. The rHuEPO produced was approximately 30 kDa. All three N-linked glycosylation sites were occupied dominantly by Man(17)(GlcNAc)(2). N-glycanase-treated rHuEPO purified but not digested with factor-Xa-protease, showed a spectral peak centered about m/z 20400 Da. The native polypeptide form of human EPO (c. 18 kDa) was obtained for the first time in P. pastoris expression system, after affinity purification, deglycosylation and factor-Xa-protease digestion. The amount of sodium dodecyl sulfate used prior to deglycosylation was found to be crucial in determining the dominant form of glycan in glycoproteins. The novel approaches to protein expression and purification system and structural analysis presented, would be important especially for therapeutic proteins expressed in P. pastoris.

Enhancement of recombinant protein production in Chinese hamster ovary cells through anti-apoptosis engineering using30Kc6 gene

It was previously reported that silkworm hemolymph (SH) inhibits apoptosis and increases the production of recombinant human erythropoietin (EPO) in Chinese hamster ovary (CHO) cells. The apoptosis-inhibiting component in SH is a member of 30K protein family. In this study, the CHO cell line producing EPO was manipulated genetically to express the 30Kc6 gene encoding a 30K protein in the hemolymph of the silkworm, Bombyx mori. The transient expression of 30Kc6 significantly suppressed the cell death induced by serum deprivation. A stable cell line expressing 30Kc6 with an anti-apoptotic property was established. The stable expression of 30Kc6 inhibited serum-deprivation-induced apoptosis and increased the cell density and EPO titer by 5- and 10-fold, respectively. The positive effects of the 30Kc6 expression on cell viability and productivity were due to the stable maintenance of the mitochondrial activity. The 30Kc6 expression efficiently suppressed the depolarization of the mitochondrial membrane and subsequently balanced the generation/consumption of ATP. The use of the 30Kc6 gene is expected to provide a new method of host cell engineering for improving the productivity of the recombinant protein.

Erythropoietin in Heart Failure and Other Cardiovascular Diseases: Hematopoietic and Pleiotropic Effects

Erythropoietin is a hypoxia-induced hormone that is a major regulator of normal erythropoiesis. Over the last decade, the production of recombinant human erythropoietin has revolutionized the treatment of anemia associated with chronic renal failure, and has led to a greater understanding of anemia pathophysiology and to the elucidation of the interactions of erythropoietin, iron, and erythropoiesis. Anemia has been shown to be independently associated with increased mortality and disease progression. Potential survival benefits associated with correction of anemia have expanded considerably the indications of erythropoietin use in various patient populations and are leading to consideration of earlier, more aggressive treatment of mild to moderate anemia. The results of such treatment are promising in a variety of new clinical settings, including anemia associated with congestive heart failure. Furthermore, the erythropoietin receptor is widely distributed in the cardiovascular system, including endothelial cells, smooth muscle cells and cardiomyocytes and preclinical studies have established erythropoietin to be a pleiotropic cytokine with anti-apoptotic activity and tissue-protective actions in the cardiovascular system, beyond correction of hemoglobin levels. Despite some potential adverse effects, such as hypertension, and the occurrence of erythropoietin resistance, early studies in heart failure patients with anemia suggest that erythropoietin therapy is safe and effective in reducing left ventricular hypertrophy, enhancing exercise performance and increasing ejection fraction. Anemia is found in about one-third of all cases of congestive heart failure (CHF). The most likely common cause is chronic renal insufficiency, which is present in about half of all CHF cases. However, anemia can occur in CHF without renal insufficiency and is likely to be due to excessive cytokine production. The anemia itself can worsen cardiac function, both because it causes cardiac stress through tachycardia and increased stroke volume, and because it can cause a reduced renal blood flow and fluid retention, adding further stress to the heart. Long-standing anemia of any cause can cause left ventricular hypertrophy, which can lead to cardiac cell death through apoptosis and worsen CHF. Therefore, a vicious circle, cardio-renal anemia syndrome, is set up wherein CHF causes anemia, and the anemia causes more CHF and both damage the kidneys worsening the anemia and the CHF further and increasing mortality. There is now evidence that early correction of the CHF anemia with subcutaneous erythropoietin and intravenous iron improves shortness of breath and fatigue, cardiac function, renal function and exercise capacity, reducing the need for hospitalization and improving quality of life. In the present review we discuss the data on current clinical use of erythropoietin in cardiovascular disease, with the main focus on the treatment of congestive heart failure, and summarize the advances and progress made in the understanding of the hematopoietic and pleiotropic effects of erythropoietin in the cardiovascular system.

Generation and Phenotypic Analysis of a Transgenic Line of Rabbits Secreting Active Recombinant Human Erythropoietin in the Milk

Production of recombinant human erythropoietin (rhEPO) for therapeutic purposes relies on its expression in selected clones of transfected mammalian cells. Alternatively, this glycoprotein can be produced by targeted secretion into the body fluid of transgenic mammals. Here, we report on the generation of a transgenic rabbits producing rhEPO in the lactating mammary gland. Transgenic individuals are viable, fertile and transmit the rhEPO gene to the offspring. Northern blot data indicated that the expression of the transgene in the mammary gland is controlled by whey acidic protien (WAP) regulatory sequences during the period of lactation. While the hybridization with total RNA revealed the expression only in the lactating mammary gland, the highly sensitive combinatory approach using RT-PCR/hybridization technique detected a minor ectopic expression. The level of rhEPO secretion in the founder female, measured in the period of lactation, varied in the range of 60-178 and 60-162 mIU/ml in the milk and blood plasma, respectively. Biological activity of the milk rhEPO was confirmed by a standard [3H]-thymidine incorporation test. Thus, we describe the model of a rhEPO-transgenic rabbit, valuable for studies of rhEPO glycosylation and function, which can be useful for the development of transgenic approaches designed for the preparation of recombinant proteins by alternative biopharmaceutical production.

The cardiovascular effects of erythropoietin.

Erythropoietin is a hypoxia-induced hormone that is essential for normal erythropoiesis. The production of recombinant human erythropoietin (rHuEpo) has revolutionized the treatment of anemia associated with chronic renal failure and chemotherapy, and has been used as prophylaxis to prevent anemia after surgery. The erythropoietin receptor is widely distributed in the cardiovascular system, including endothelial cells, smooth muscle cells and cardiomyocytes. Epo has potentially beneficial effects on the endothelium including anti-apoptotic, mitogenic and angiogenic activities. On the other hand, some reports suggest that rHuEpo may have pro-thrombotic or platelet-activating effects. Hypertension develops in 20-30% of renal patients treated with rHuEpo. Many patients with heart failure have anemia. Despite some potential adverse effects, early studies in heart failure patients with anemia suggest that rHuEpo therapy is safe and effective in reducing left ventricular hypertrophy, enhancing exercise performance and increasing ejection fraction. Further studies are warranted to define the role of rHuEpo in chronic heart failure and other cardiovascular settings.

Advances in anaemia management: Current evidence

SUMMARY: Simplicity and sufficiency in the management of renal anaemia were first envisaged with the development and production of recombinant human erythropoietin (epoetin). Recent years, however, have seen the publication of several studies examining the optimal administration of epoetin, the emergence of a new erythropoietic agent (Novel Erythropoietic Stimulating Protein, NESP), recognition of the need for supraphysiological iron levels, and the possible use of many potential adjuvant agents. Together, these factors have made the management of renal anaemia substantially more complex, perhaps even to the point of confusion. the purpose of this review, therefore, is to examine the evidence relating to these factors in order to offer a summary of their known effects on anaemia management, hopefully with some progress towards the initial goals.

Enhancement of recombinant erythropoietin production in CHO cells in an incubator without CO(2) addition.

The effect of low levels of carbon dioxide (CO(2)) in the gas phase on the production of recombinant human erythropoietin (EPO)in CHO cells was explored. A T-flask culture in an incubator without CO(2) addition showed a slow cell growth initially followed by the cessation of growth, while other cultures incubated under 0.5-5% CO(2) concentrations grew normally at the same rate during the entire period of cultivation. Interestingly, the production of EPO in the culture incubated under no CO(2) supply was highest among the tested cultures. The cell specific secretion rate of EPO (q(EPO)) of the culture under no CO(2) supply was about 3 times higher than that of the culture under 5% CO(2) supply. Western blot analysis and in vivo bioassay of EPO showed no apparent changes in EPO quality between the two cases of different CO(2) environments (air vs. 5% CO(2)), suggesting robust glycosylation of EPO by CHO cells even under very reduced CO(2) environment. Various combinations of the two extreme cases, with 5% CO(2) supply (suitable for cell growth) and no CO(2) addition (better for EPO production), were made in order to maximize the volumetric productivity of EPO secretion (P(V)) in CHO cells. The P(V) of the cultures programmed with initial incubation under 5% CO(2) followed by no CO(2) supply was about 2 times superior to that of the culture incubated only under no CO(2) supply. The P(V) of the culture under no CO(2) supply was slightly lower than that of culture grown under 5% CO(2). However, the q(EPO) of the no CO(2) supply case was more than 5 times higher than that of the culture under 5% CO(2) supply. In conclusion, we have demonstrated that a simple programming of CO(2) supply to an incubator can enhance the production of EPO in CHO cells remarkably, without any apparent change of the EPO quality.

Efficient production of recombinant human erythropoietin by replenishment of microcarriers in the hollow fiber culture cassette.

Human erythropoietin (EPO)-producing recombinant BHK cells were cultured in culture medium containing microcarriers, and then microcarriers attached with cells were replenished in the hollow fiber culture cassette. By culture for 14 days, it was possible to produce 450 micrograms of the recombinant EPO, which corresponded to over two-fold of the recombinant EPO production by control hollow fiber culture without microcarriers.