Discovery and basic pharmacology of erythropoiesis-stimulating agents (ESAs)'including the hyperglycosylated ESA, darbepoetin alfa: An update of the rationale and clinical impact

Amgen Kft., Budapest, Hungary.
European Journal of Clinical Pharmacology (Impact Factor: 2.97). 04/2010; 66(4):331-40. DOI: 10.1007/s00228-009-0780-y
Source: PubMed


Cloning of the human erythropoietin (EPO) gene and development of the first recombinant human erythropoietin (rHuEPO) drug were truly breakthroughs. This allowed a deeper understanding of the structure and pharmacology of rHuEpo, which in turn inspired the discovery and development of additional erythropoiesis-stimulating agents (ESAs). In vivo specific activity and serum half-life of rHuEPO are influenced by the amount and structure of the attached carbohydrate. Increased numbers of sialic acids on carbohydrate attached to rHuEPO correlated with a relative increase in in-vivo-specific activity and increased serum half-life. The effect of increasing the number of sialic-acid-containing carbohydrates on in-vivo-specific activity was explored. Initial research focused on solving the problem of how the protein backbone could be engineered so a cell would add more carbohydrate to it. Additional work resulted in darbepoetin alfa, a longer-acting molecule with two additional carbohydrate chains.

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    • "It supports the production of red blood cells (Durocher and Butler, 2009; Goldwasser, 1975). Despite many efforts to synthesize this protein in many different expression systems, production of recombinant human EPO (rHuEPO) still relies on chinese hamster ovary (CHO) derived cells (Kiss et al., 2010). Endogenous human EPO EPO is a 30- 38kDa glycoprotein (exact molecular weight depends on the degree of glycosylation) in which 60% of the molecule composed of 165 amino acid single polypeptide chain containing two disulphide bonds. "

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    • "The process of glycosylation, unlike the synthesis of amino acids and nucleic acids, is enzymatic rather than defined by a template, and hence the carbohydrates are diverse with respect to both the number and the linkage patterns of the sugar units.25 Indeed, the carbohydrate composition of rHuEPO N-linked chains is complex and differs in terminal N-acetylneuraminic acid (Neu5Ac) content, O-acetylation of the Neu5Ac residues, N-acetylactosamine extensions, and degree of branching.26,27 Thus, changes in cell lines, growth conditions, or manufacturing processes can affect the final rHuEPO characteristics, including the generation of microheterogeneity in glycosylation.28 Carbohydrate addition to proteins can influence many aspects of a protein’s properties including molecular stability, solubility, immunogenicity, and in vitro and in vivo biological activity. "
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    ABSTRACT: Recombinant human erythropoietin (rHuEPO), such as the approved agents epoetin alfa and epoetin beta, has been used successfully for over 20 years to treat anemia in millions of patients. However, due to the relatively short half-life of the molecule (approximately 8 hours), frequent dosing may be required to achieve required hemoglobin levels. Therefore, a need was identified in some anemic patient populations for erythropoiesis stimulating agents with longer half-lives that required less frequent dosing. This need led to the development of second generation molecules which are modified versions of rHuEPO with improved pharma-cokinetic and pharmacodynamic properties such as darbepoetin alfa, a hyperglycosylated analog of rHuEPO, and pegzyrepoetin, a pegylated rHuEPO. Third generation molecules, such as peginesatide, which are peptide mimetics that have no sequence homology to rHuEPO have also recently been developed. The various molecular, pharmacokinetic, and pharmacodynamic properties of these and other erythropoiesis stimulating agents will be discussed in this review.
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    • "These can be analyzed by a variety of analytical methods (Butler and Perreault 2010), some of which have become high throughput (Ruhaak et al. 2010). Enhanced sialylation of recombinant erythropoietin by the introduction of two extra glycan sites results in a hyperglycosylated protein (darbepoetin) with a significantly greater serum half-life and considerable clinical advantages (Kiss et al. 2010). For immunoglobulin antibodies, it is clear that alterations of glycoforms occur under physiological and pathological conditions in vivo. "
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    ABSTRACT: The demand for production of glycoproteins from mammalian cell culture continues with an increased number of approvals as biopharmaceuticals for the treatment of unmet medical needs. This is particularly the case for humanized monoclonal antibodies which are the largest and fastest growing class of therapeutic pharmaceuticals. This demand has fostered efforts to improve the efficiency of production as well as to address the quality of the final product. Chinese hamster ovary cells are the predominant hosts for stable transfection and high efficiency production on a large scale. Specific productivity of recombinant glycoproteins from these cells can be expected to be above 50 pg/cell/day giving rise to culture systems with titers of around 5 g/L if appropriate fed-batch systems are employed. Cell engineering can delay the onset of programmed cell death to ensure prolonged maintenance of productive viable cells. The clinical efficacy and quality of the final product can be improved by strategic metabolic engineering. The best example of this is the targeted production of afucosylated antibodies with enhanced antibody-dependent cell cytotoxicity, an important function for use in cancer therapies. The development of culture media from non-animal sources continues and is important to ensure products of consistent quality and without the potential danger of contamination. Process efficiencies may also be improved by employing disposable bioreactors with the associated minimization of downtime. Finally, advances in downstream processing are needed to handle the increased supply of product from the bioreactor but maintaining the high purity demanded of these biopharmaceuticals.
    Full-text · Article · Oct 2012 · Applied Microbiology and Biotechnology
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