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Recombinant EPO production–points the nephrologist should know Wolfgang Jelkmann Erythropoetin • Glycoprotein hormone that controls erythropoesis • Produced by the peritubular capillary endothelial cells in the kidney rhEPO • Provides enormous benefit in the prevention and reversal of anemia in chronic kidney disease, malignancy and AIDS • Supports autologous blood collection Source: http://www.sciprogen.com/en/products111.html rhEPO-type substances • Represent the largest market of a class of biopharmaceuticals • Global estimated sales of ~1010 euro (Php 600 x 1010) per annum EPO Formation • rhEPO is produced with the use of cells transfected with either the human EPO gene or EPO cDNA • Present therapeutic rhEPO preparations are manufactured in mammalian host cells • EPO is a complex glycoprotein of 165 amino acids to which four glycans are attached EPO Formation Addition of sugar molecules to dolichol ↓ Glycan transferred to growing polypeptide ↓ Folded and moved to Golgi complex ↓ N-acetylglucosamine, galactose and sialic acid (Nacetylneuraminic acid) finally added Nomenclature • Stem: “-poetin” • Epoietin: eucaryotic cell-derived rhEPO • Changes in the amino acid sequence indicated by a different prefix (eg: darbepoetin) • Analogues with an altered glycosylation pattern due to production in a different host cell system classified by a Greek letter added to the name (eg: epoetin-ω, epoetin-α) Nomenclature • Epoetins possess 3 tetra-antennary N-linked (Asn 24, 38 and 83) and 1 small O-linked (Ser 126) glycans • Carbohydrates amount to 40% of the total molecular mass of EPO (30.4 kDa) • Survival of EPO in circulation requires the presence of terminal sialic acid residues of its Nglycans • Asialo-glycoproteins are rapidly cleared via galactose-receptors of hepatocytes Differences between Epoetins • Chinese hamster ovary (CHO) cells deficient in the dihydrofolate reductase gene are most commonly used for the large-scale pharmaceutical manufacture of glycoproteins • CHO cell-derived products: – Epoetin-α (Epogen, Procrit, Eprex, Erypo, Espo) – Epoetin-β (Recormon, NeoRecormon, Epogin) Differences between Epoetins • Epoetin-α is more homogenous and possesses less basic isoforms than epoetin-β • Plasma half-life of epoetin-α was measured to be shorter than that of epoetin-β by some investigators • The two established products are clinically equivalent • Transiently higher incidence of antibody formation towards an epoetin-α formulation (Eprex) Safety Considerations • The clinical efficacy and safety of biosimilar rhEPO preparations should be demonstrated in at least two adequately powered, randomized, parallel group clinical trials in comparison to a reference product • The transgene, the host cell line, the culture conditions and the purification procedures applied by a follow-on manufacturer cannot be the same as the original • The manufacturing process cannot be exactly copied Safety Considerations • Epoetin-ω (Epomax, Hemax): Expressed in baby hamster kidney cells – Amino acid sequence is unaltered – Has an N-glycan with phosphorylated oligomannoside chains and it possesses less O-glycans Safety Considerations • Epoetin-δ: expressed in the human fibrosarcoma cell line—HT 1080 derivative (others in lymphoblastoid RPMI 1788 cells) – Possesses less N-glycolylneuraminic acid residues (Neu5Gc) than CHO cell-derived epoetins EPO Congeners • Darbepoetin-α (Aranesp) – rhEPO mutein (a product with altered amino acid sequence) – increased molecular mass (37.1 kDa vs 30.4 kDa) and increased proportion of carbohydrate (51% vs 40%) – Lower affinity for the EPO receptor (EPO-R) but a longer survival in circulation – Terminal half-life: 3-4x lonnger than that of epoetin-α or epoetin-β (25 vs 6–9 h) EPO Congeners • Pegylated epoetin-β (CERA, ‘continuous erythropoiesis receptor activator’) – Longer half-life (130–140 h) than darbepoetinα – Contains a single methoxypolyethylene glycol (PEG) polymer of 30 kDa – Prolonged survival in the circulation due to large molecular mass (60 kDa) and low EPOR binding affinity EPO Congeners • Hematide – A synthetic (non-recombinant) dimeric EPO mimetic peptide linked to PEG – Induces reticulocytosis and increases hemoglobin levels in healthy volunteers in phase I studies Conclusion • Improve the efficacy and pharmacokinetics of the drugs and to reduce their costs • Factors to consider: plasmids, type of promoter, marker genes, transformed host cell, production process, purification steps, posttranslational modifications, way of formulation, and immunogenicity • Safety of novel biopharmaceuticals can be proven only by clinical experience