Macromolecule ADME + Recombinant Proteins Flashcards

1
Q

How is HIF-1α normally degraded?

A

Hydroxylated HIF-1α is recognised and targetted for ubiquitination, which is then degraded by 26s proteasome (ubiquitin-proteasome system)

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2
Q

What happens in Von Hippel-Lindau disease?

A

pVHL cannot degrade HIF-1α, increasing VEGF, migration, invasion, metastasis and angiogenesis, predisposing to highly vascularised tumours

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3
Q

What are the two means of protein degradation in mammalian cells?

A

Lysosomal degradation (10%)
Proteolysis (90%)

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4
Q

The proteasome in mammalian cells is the ___S proteasome

A

26s

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5
Q

The proteasome is made up of a ___s degradation chamber with two ___s caps, and a channel that is ___Å wide

A

20s, 19s, 13Å

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6
Q

What is attached to the 19s subunit caps?

A

ATP subunits (energy dependent)

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7
Q

What does ubiquitin do for proteolysis

A

Polyubiquitin tag (at least 4 x Ub) acts as a label for the 19s cap to recognise and bind to

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8
Q

What enzyme cleaves the polyubiquitin tag?

A

deubiquitinising enzymes (DUB)

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9
Q

What are the 3 methods by which proteins can be delivered to the proteasome?

A
  1. binding directly to the 19s subunit
  2. brought to the proteasome by 3rd party adaptor proteins
  3. degraded without ubiquitin
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10
Q

What are the 5 challenges of BCPs?

A
  1. Immunogenecity (CHO cell pdts if not well purified)
  2. Susceptibility to denaturation (leak out from cell to ECF)
  3. If MW > 200kDa, eliminated via phagocytosis
  4. Susceptible to degradation in cells by lysosomes and proteasomes
  5. Distribution limited by permeability and porosity of the vasculature
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11
Q

What are the two main methods of movement after administration?

A

Diffusion and convection (bulk movement with fluid)

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12
Q

Where do large proteins > 16-20 kDa enter?

A

Lymph mainly

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13
Q

Where do small proteins < 16-20 kDa enter?

A

Both lymph and blood

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14
Q

What are the 2 main factors that affect distribution?

A

Size/MW and protein binding

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15
Q

How are macromolecules usually degraded?

A

Proteolysis

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16
Q

Which two biological molecules do FcRn bind to?

A

serum albumin and IgG

17
Q

What does FnRn binding help the biological molecules to do?

A

Escape lysosomal degradation

18
Q

What factor influences FnRn binding?

A

pH (low pH bind, neutral pH dissociate)

19
Q

What 4 factors affect elimination?

A
  1. MW
  2. Charge (+ve excreted more as glomerular membrane is -ve)
  3. Shape and rigidity
  4. Tubular reabsorption (+ve reabsorbed more as membrane is -ve)
20
Q

What are 3 strategies to improve PK properties?

A
  1. Glycosylation
  2. PEGylation (attached to Lys)
  3. Increase MW by fusion proteins (fuse w Fc domains)
21
Q

When can proteins lose biological activity? (3)

A
  1. Protein recovery from source
  2. Protein purification process
  3. Post protein storage
22
Q

What are the 3 states that proteins can exist in?

A

Native, Unfolded, Aggregated

23
Q

What is the main force causing aggregation?

A

Hydrophobic interactions

24
Q

What are the 7 physical factors that affect protein stability?

A
  1. Temperature
  2. pH (extremes mess w ionisation)
  3. Adsorption (affect secondary and tertiary structures)
  4. Shaking and shearing (air bubble interface
  5. Non-aqueous solvents (disrupts polarity)
  6. Repeated freeze-thaw
  7. Photodegradation (esp Tyr)
25
What are the 4 chemical factors that affect protein stability?
1. Deamination (Asn, Gln) 2. Oxidation (His, Met, Cys, Try, Tyr) 3. Disulfide bond formation or breakage (Cys) 4. Hydrolysis (Asn-Gly and Asn-Pro are susceptible)
26
What are 4 methods of chemical modification to stabilise liquid protein solutions?
1. AA substitution or modification 2. Introduction of disulfide bonds (risk of folding diff) 3. PEGylation (increase circulation time) 4. Acylation (lipophilic, repels water)
27
How does recombinant DNA (rDNA) affect BCP? (4)
1. overcomes source availability 2. allows safer production 3. provides alternative method (no inappropriate sources) 4. offers chance to design desirable mutations