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 (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