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Biotherapeutics > EPO > Flashcards

EPO Flashcards

1
Q

What are 4 structural characteristics of EPO?

A

165 aa
2 disulfide bonds
3 N-glycosylation sites
1 O-glycolystaion site

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2
Q

What does EPO stimulate?

A

Survival, proliferation, differentiation of erthryocyte precursors

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3
Q

Where is EPO produced in adults?

A

Kidneys in peritubular fibroblasts in renal cortex

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4
Q

Where is EPO produced in the fetus?

A

Liver

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5
Q

How does hypoxia cause EPO production?

A

HIF-1B/HIF-2a binds to HRE

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6
Q

What are the 2 proline residues found in HIF2a C terminus?

A

Pro405
Pro531

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7
Q

What are the proline residues hydroxylated by?

A

alpha ketoglutarate requiring dixoygenases in O2

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8
Q

What is HIF’s asparagine residue hydroxylated by?

A

FIH

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9
Q

What does EPO promote in bone marrow?

A

Proliferation & differentiation of erythrocytic progenitors like CFU-Es)

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10
Q

What are characteristics of EpoR?

A

59 kDA glycoprotein (484 aa & 1 N-glycan) that acts as a homodimer

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11
Q

What does Epo binding cause?

A

Intracellular activation of EpoR-associated JAK-2 -> tyrosine phosphorylation

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12
Q

What receptor family is EpoR a part of?

A

type 1 cytokine receptor

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13
Q

What is signal transduction through EpoR intiated by?

A

Ligand binding -> dimerization/reorientation of EpoR monomers

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14
Q

Explain the steps following EpoR activation?

A

JAK2 phosphorylates Tyr residues in EpoR -> docking sites for signalling molecules with phosphotyrosine binding motifs -> STAT5

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15
Q

When is the action of EPO terminated?

A

When EpoR is dephosphorylated by SHP-1 -> complex is internalized

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16
Q

What is recombinant human EPO used for?

A

Treatment of different types of anemia

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17
Q

What do Epoietins have to be produced in and why?

A

mammalian cells (CHO) as EPO is a glycoprotein

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18
Q

What is a structural difference in Epoietins to endogenous EPO?

A

glycans have minor differences

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19
Q

What 3 things influence glycosylation?

A

host cell species & clone
culturing process
purification steps

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20
Q

What does the major N-glycan structure of urinary hEPO consist of?

A

fucose-containing sialylated tetra-antennary glycan

21
Q

What is the in-vivo half-life of glycoproteins determined by?

A

Sialic acid capping in terminal sugar on N-linked glycans

22
Q

What is responsible for the biological activity of rhEPO in vivo?

A

Terminal sialylation level

23
Q

What do sialylations mask?

A

Penultimate galactose residues -> captured by asialoglycoprotein receptors in hepatocytes -> degradation

24
Q

What are 3 pros of glycosylations?

A

Protect protein from proteolysis
enhance overall solubilty
modulate receptor binding

25
Q

How is glycosylation optimized?

A

Engineering of EPO
Engineering of production hosts
culture condition optimization

26
Q

What’s an example of hyperglcosylation?

A

Darbepoetin alfa endowed with 2 extra N-glycans -> 22 sialic acid residues -> increase in plasma half-life & enhanced in vivo activity

27
Q

Whats an example of PEGylation?

A

ehEPO to PEG -> CERA -> half life of 130hrs

28
Q

What are 3 target points for enhanced sialylation?

A

IC level of nucleotide sugar CMP-sialic acid
transportation of CMP-sialic acid to Golgi complex
sialylation of glycans

29
Q

What are 3 modes of culture production?

A

batch
fed-batch
perfusion

30
Q

What is used for large-scale production of recombined glycoproteins?

A

fed-batch & perfusion

31
Q

What can affect the culture performance & glycosylation profile?

A

culture temp
pH

32
Q

What does high conc. of ammonia alter?

A

Glycosylation pattern of rhEPO by decreasing terminal sialylation, content of O-linked glycan & tetra-antennary of N-glycan

33
Q

What affects the incidence & degree of immunogenicity in Epoietins?

A

product-specific & patient specific parameters

34
Q

What are 5 modification in place of glycans in SEP?

A

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

35
Q

What are 3 yeilds of SEP?

A

solid phase peptide synthesis 15-30%
oximation 30-50%
NCL 40-70%
over 100mg of SEP obtained

36
Q

What is SPPS?

A

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

37
Q

What are 3 benefits of using E.coli for EPO engineering?

A

High protei yield
low cost
no post-translational glycosylation
however non-glycosylated proteins are less stable & prone to aggregation

38
Q

What is used for chem-selective conjugation of defined carbohydrate moieties?

A

copper-catalyzed azidde-alkyne cycloaddition

39
Q

What was found after Plk was introduced to glycosylated EPO variants?

A

Biologically active
successful induction of differentiation into erythrocyte precursor
glycans remain intact after CuAAC

40
Q

What are 5 advantages of the Staudinger Phosphite Reaction?

A

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

41
Q

What does a single short-branched-PEG chain enhance?

A

resistance against proteolysis
unsepcific aggregation

42
Q

What are structural features of human interferon-gamma?

A

143 aa
dimeric structure
no disulfide bonds

43
Q

What is human interferon gamma used for?

A

critical for innate & adaptive immunity
approved for chronic granulomatous disease & malignant osteoetrosis
anticancer applications

44
Q

What happens after ligandbing to IFN-yR?

A

JAk2 autophosphorylated -> activated -> JAk1 transphorylation

45
Q

What are 3 signalling molecules activated by IFN-y?

A

ICAM-1
MIG
iNOS

46
Q

What is used for semi-synthesis of homogenous hIFy glycocongugates and why?

A

PoxF -> high protein yields & reproducible incorporation experiments

47
Q

What are the 6 steps in semi-synthesis?

A

recovery from inclusion bodies
affinity chromotography
refolding
size exclusion chromotography
CuAAc
size exclusion chromotography

48
Q

What was the result of using the nnAA PoxF?

A

too disruptive for scaffold of hIFy
triazole linker formed after CuAAC is not tolerated
cytokines are not robust model proteins

Biotherapeutics (9 decks)

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