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

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rhEPO-type substances Represent the largest market of a class of biopharmaceuticals Global estimated sales of ~10 10 euro (Php 600 x ) 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 (N- acetylneuraminic 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 N- glycans 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 darbepoetin- α –Contains a single methoxypolyethylene glycol (PEG) polymer of 30 kDa –Prolonged survival in the circulation due to large molecular mass (60 kDa) and low EPO- R 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