EPO Flashcards
What are 4 structural characteristics of EPO?
165 aa
2 disulfide bonds
3 N-glycosylation sites
1 O-glycolystaion site
What does EPO stimulate?
Survival, proliferation, differentiation of erthryocyte precursors
Where is EPO produced in adults?
Kidneys in peritubular fibroblasts in renal cortex
Where is EPO produced in the fetus?
Liver
How does hypoxia cause EPO production?
HIF-1B/HIF-2a binds to HRE
What are the 2 proline residues found in HIF2a C terminus?
Pro405
Pro531
What are the proline residues hydroxylated by?
alpha ketoglutarate requiring dixoygenases in O2
What is HIF’s asparagine residue hydroxylated by?
FIH
What does EPO promote in bone marrow?
Proliferation & differentiation of erythrocytic progenitors like CFU-Es)
What are characteristics of EpoR?
59 kDA glycoprotein (484 aa & 1 N-glycan) that acts as a homodimer
What does Epo binding cause?
Intracellular activation of EpoR-associated JAK-2 -> tyrosine phosphorylation
What receptor family is EpoR a part of?
type 1 cytokine receptor
What is signal transduction through EpoR intiated by?
Ligand binding -> dimerization/reorientation of EpoR monomers
Explain the steps following EpoR activation?
JAK2 phosphorylates Tyr residues in EpoR -> docking sites for signalling molecules with phosphotyrosine binding motifs -> STAT5
When is the action of EPO terminated?
When EpoR is dephosphorylated by SHP-1 -> complex is internalized
What is recombinant human EPO used for?
Treatment of different types of anemia
What do Epoietins have to be produced in and why?
mammalian cells (CHO) as EPO is a glycoprotein
What is a structural difference in Epoietins to endogenous EPO?
glycans have minor differences
What 3 things influence glycosylation?
host cell species & clone
culturing process
purification steps
What does the major N-glycan structure of urinary hEPO consist of?
fucose-containing sialylated tetra-antennary glycan
What is the in-vivo half-life of glycoproteins determined by?
Sialic acid capping in terminal sugar on N-linked glycans
What is responsible for the biological activity of rhEPO in vivo?
Terminal sialylation level
What do sialylations mask?
Penultimate galactose residues -> captured by asialoglycoprotein receptors in hepatocytes -> degradation
What are 3 pros of glycosylations?
Protect protein from proteolysis
enhance overall solubilty
modulate receptor binding
How is glycosylation optimized?
Engineering of EPO
Engineering of production hosts
culture condition optimization
What’s an example of hyperglcosylation?
Darbepoetin alfa endowed with 2 extra N-glycans -> 22 sialic acid residues -> increase in plasma half-life & enhanced in vivo activity
Whats an example of PEGylation?
ehEPO to PEG -> CERA -> half life of 130hrs
What are 3 target points for enhanced sialylation?
IC level of nucleotide sugar CMP-sialic acid
transportation of CMP-sialic acid to Golgi complex
sialylation of glycans
What are 3 modes of culture production?
batch
fed-batch
perfusion
What is used for large-scale production of recombined glycoproteins?
fed-batch & perfusion
What can affect the culture performance & glycosylation profile?
culture temp
pH
What does high conc. of ammonia alter?
Glycosylation pattern of rhEPO by decreasing terminal sialylation, content of O-linked glycan & tetra-antennary of N-glycan
What affects the incidence & degree of immunogenicity in Epoietins?
product-specific & patient specific parameters
What are 5 modification in place of glycans in SEP?
chemoselective linker
hydrophilic spacer consisting of 1 polymer repat unit
core structure with 4 branch points
linear polymer with 12 repeat units attached to each branch point
-ve charge-control unit at end of each linear polymer
What are 3 yeilds of SEP?
solid phase peptide synthesis 15-30%
oximation 30-50%
NCL 40-70%
over 100mg of SEP obtained
What is SPPS?
stepwise assembly based on repetitive process resulting in elongation of a polymer-bound peptide chain by successive aa additions until peptide of desired sequence & length has been synthesized
What are 3 benefits of using E.coli for EPO engineering?
High protei yield
low cost
no post-translational glycosylation
however non-glycosylated proteins are less stable & prone to aggregation
What is used for chem-selective conjugation of defined carbohydrate moieties?
copper-catalyzed azidde-alkyne cycloaddition
What was found after Plk was introduced to glycosylated EPO variants?
Biologically active
successful induction of differentiation into erythrocyte precursor
glycans remain intact after CuAAC
What are 5 advantages of the Staudinger Phosphite Reaction?
site-specific modification
simple reaction without toxic additives
tolerant against denaturing salts & high temps
can be performed in semi-purified samples
results in coupling of branched PEG -> improved protection
What does a single short-branched-PEG chain enhance?
resistance against proteolysis
unsepcific aggregation
What are structural features of human interferon-gamma?
143 aa
dimeric structure
no disulfide bonds
What is human interferon gamma used for?
critical for innate & adaptive immunity
approved for chronic granulomatous disease & malignant osteoetrosis
anticancer applications
What happens after ligandbing to IFN-yR?
JAk2 autophosphorylated -> activated -> JAk1 transphorylation
What are 3 signalling molecules activated by IFN-y?
ICAM-1
MIG
iNOS
What is used for semi-synthesis of homogenous hIFy glycocongugates and why?
PoxF -> high protein yields & reproducible incorporation experiments
What are the 6 steps in semi-synthesis?
recovery from inclusion bodies
affinity chromotography
refolding
size exclusion chromotography
CuAAc
size exclusion chromotography
What was the result of using the nnAA PoxF?
too disruptive for scaffold of hIFy
triazole linker formed after CuAAC is not tolerated
cytokines are not robust model proteins
