Recombinant EPO production–points the nephrologist should know Wolfgang Jelkmann
Jan 01, 2016
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