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Erythropoietin (EPO) is obligatory growth factor (hormone) in many mammalians by promoting the formation of new red blood cells. In the body, EPO is produced mainly in kidney, circulated to bone marrow where it activates the proliferation and differentiation of erythroid progenitors. During last two decades, recombinant human erythropoietin protein (rhEPO) has become the most successful drug produced by recombinant DNA technology. Millions of people worldwide have benefited from research on erythropoietin. Epoetin alfa (EPO-¡), a rhEPO trade mark from Amgen, has been widely used to correct anemia. Fatigue, weakness, and shortness of breath are common symptoms seen in anemic patients due to the lack of oxygen carrying to target tissues. In many types of cancer, tumor hypoxia is associated with poor prognosis and chemo- or radio-therapy resistance. Besides, rhEPO is an effective way to reduce the risks in blood transfusion. Therefore patients with chronic kidney decease, hemalotogic disorders, or cancer-related anemia are most benefit from rhEPO.
Table 1.1: Milestones in studying of erythropoietin [77-78]
Bert describes the first time the relationship between increase red blood cells in people living in high attitudes consequently increased blood O2 capacity.
Carnot et al. rEPO report a humoral hormone responsible for red blood cell production which then is known as erythropoietin
Researchers give evidences for increase of young red blood cells (reticulocytes) when is exposed to hypoxic plasma factor.
Reissman et al. shows on parabiotic rats that the induction of erythroid hyperplasia in bone marrow and production of reticulocytes in both parabiotic rats when only one partner is exposed to hypoxic condition.
Erslev et at. confirms the increase of reticulocytes and hematocrit in rabbits which were subjected with plasma from anemic donor animals.
Jacobson et al. find out that erythropoietin is mainly produced at the kidney.
Zanjani at al. show liver is the main site for EPO production in the fetus. However, Fried figure out a small amount of EPO is still synthesized at the liver after birth
Miyake et al. purify human erythropoietin from the urine of anemic patients.
Two independent research groups leading by Lin and Jacobs successful clone and express the human erythropoietin gene in Chinese hamster ovary and green monkey kidney cell, respectively.
Eschbach and colleagues first use recombinant human erythropoietin to correct the anemia of chronic renal disease.
The FDA approve for using recombinant human erythropoietin for the treatment anemia of chronic renal failure.
The FDA approve for using recombinant human erythropoietin for cancer-related anemia.
1.4.2 Structure of erythropoietin
EPO is a glycoprotein hormone belonging to the class I cytokine family. The human EPO gene locates in chromosome 7, which is about 2.2 kb, encoding for 193 amino acids that include a 27 amino acid signal peptide and 166 core amino acids linked with 4 carbohydrate side chains (account for 40% molecular weight) which are the critical structures for half life of this protein in vivo. Its molecular mass is 30.4 kDa  which tertiary structure folds as a bundle of globular form with 4 alpha helices .Three of four carbohydrate side chains, N-linked oligosaccharides at asparagines 24, 38 and 83, are important for protein stabilization and activity. However, the O-linked oligosaccharide at serine 126 appears to lack of functional importance . The mature hEPO has a half-life of about 8 hours in blood circulation.
Fig. 1.3 Amino acid sequence (A) and predicted tertiary structure (B) of human EPO. (A) Primary sequence of EPO contains 165-166 aa, including 3 N-linked and 1 O-linked glycosylation. The site of O-glycosylation is represented by an open hexagon; sites of N-glycosylation are represented by filled hexagons. Rectangles show the predicted a-helical sequences. Dashed lines indicate disulfide bonds. (B) The predicted tertiary structure of human EPO is based on folded, antiparallel arrangements of a-helices. Glycosylation sites are designated as in (A). ¡-helices are labeled A through D in order from the N-terminus to the C-terminus with intervening loops shown with an arrow directed toward the C-terminus .
Recombinant human erythropoietin
In 1985, the human EPO gene was independently cloned by two research groups within a few weeks of each other [83-84]. The hEPO primary structure consists of 166 amino acids, however, there are some debates regarding on the existing of arginine 166 (R166) in mature form. Although , By using peptide mapping and fast atom bombardment mass spectrometry, Recny and his colleagues did not see the existence of R166 in recombinant human protein purified from Chinese hamster ovary (CHO) cells , Lai al al. demonstrated the presence of R166 in human urinary EPO by protein sequencing determination . It is possible that the presence or absence of R166 depends on the cells in which it was produced or the missing is just happened during EPO circulation .
Human urinary EPO and rhEPO are identical in primary and secondary structures. However, there are minor quantitative differences in O-linked and N-linked glycan composition which make some of them distinguishable under isoelectric focusing. The species, tissues where protein is produced or the ways of protein purification will result in the difference in isoelectric patterns which is majorly due to post-translation modification . There are more than six different rhEPO forms produced in the world but only available in certain country markets . Three common uses which were approved by the FDA are EPO-¡ and EPO-¢ (recombinant form) and darbopoetin-¡ (synthetic form). They all share the similar primary protein sequences, but differ in carbohydrate side chains. In contrast to recombinant forms (EPO-¡ and EPO-¢), which have 3 N-linked carbohydrate chains with maximum 14 sialic acids, darbopoetin-¡ has 5 N-linked carbohydrate side chains and contains up to 22 sialic acids. Those differences can be an explanation for the greater half life (~25 hours) and stronger in vivo bioactivity of darbopoetin-¡ compared to recombinant ones . Isoelectric patterns from human urine have about 10 isoforms, with pIs ranging from 3.77 to 4.77, while recombinant human EPO-¡ and EPO-¢ consist of 6 and 7 isoforms (pI: 4.42 - 5.11), respectively . Both EPO-¡ and EPO-¢ showed very similar isoform pattern, although EPO-¢ has an extra basic band due to more sialic acids in carbohydrate chains . The migration patterns of rhEPO and darboetin alfa differ greatly (Fig. 1.4). Darbepoetin-¡ appeared in the basic area (anode), while rhEPO appeared in the acedic area (cathode) .