Transcript Recombinant EPO production–points the nephrologist should know

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