biologics part 2 Flashcards

1
Q

which amino acids are most likely to be buried within the protein?

A

the non-polar ones as they want to avoid contact with water HYDROPHOBIC

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2
Q

how should mAbs be conformed/

A

hydrophobic should be buried in the protein core and the hydrophilici amino acids should be in the shell

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3
Q

what do salt bridges do:?

A

they solidify the molecule but it can still move

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4
Q

what happens if the protien unfolds?

A

this may lead to aggregation if you expose the hydrophobic core then it will try and avoid contact with water. it cannot refold so there are now two proteins with hydrophobic regions exposed

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5
Q

what do you need to consider for stability of a mAb?

A

they are NOT colloids - therefore there is not a unifrom distrubution of the same charge

  • you need to consider attractive and repulsive forces
  • consider pH, buffer, salt and co-solutes
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6
Q

what may lead to rhe unfolding of the protein?

A

changes to

  • ph
  • temp
  • pressure
  • conc of co solutes
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7
Q

what happens if it undergoes chemical degradation?

A

▫ Oxidation, deamidation, hydrolysis
▫ May lead to instability then aggregation
- Exposure hydrophobic regions – which will try and avoid contact with water
-Exposure of cysteine residues of formation of disulfide bridges

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8
Q

what factor induce conformational change in shape and decrease stability?

A

▫ Extremes of pH
▫ Shear force – apply high pressure to the syringe and this is the shear force
▫ Air/water interfaces (rapid agitation, stirring and shaking)
▫ Adsorption to surfaces – need to be careful on which materials you use to ensure you don’t have adsorption on the surface
▫ Freezing, drying and re-hydration
▫ Elevated temperatures – proteins have a melting point between 40-50 degrees, so if you go towards these the protein will begin to unfold and form aggregates
▫ High pressures -these may be issues in hospital as you may now have to make very high concentrations (MORE VISCOUS) therefore if you need to inject it you will have to apply a very high pressure – this can be an issue

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9
Q

what is preferential exclusion/

A

PROTECTANT
 Lower interaction with protein but not hydrophobic leads to higher concentration of co-solute in bulk than in the solvation shell of the protein therefore not hydrophobic.
 Attract water and render surface so it makes it shrink on itself, so it is tighter and less likely to unfold

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10
Q

what is preferential interaction

A

DENATURANT
 Interaction with backbone of protein e.g. urea H-bonding with most AA side chains
 Examples: urea or guanidine hydrochloride, bind to surface of protein and it unfolds the protein.
 Within the shell and will cause an unfolding of the protein

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11
Q

how is stability testing defined?

A

ICHQ5C

international conference for harmonisation qualification of the world

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12
Q

how is shelf life determined?

A

during long term stability real time and real temperature data
- this costs alot of money and therefore needs a big company to do it

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13
Q

what are the accerlated studies?

A

▫ Support to establish the shelf life – need to keep this in line with long term stability
▫ Provide info on changes, validation of stability tests to try and relay this to what happens in long time storage
▫ Generate help to understand degradation profiles
▫ Test conditions are normally done earlier than real storage conditions

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14
Q

what are stress studies?

A

▫ Representative accidental exposures – something that happens during the process which are common, such as shaking.
▫ Reveal patterns of degradation

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15
Q

what are the issues with freezing?

A
  • Low temperature extends shelf life of medicine but cold denaturation which happens when freezing sample may lead to damage of what you have as freezing results in change of pH, ionisation, solubility or H-bond energies.
  • Repeated freezing and thawing cause aggregation by pH and concentration changes and by provision of nucleation points at ice water interface. This is a serious issue
  • Cryoprotection by sugars, polyhydric alcohols, AAs, work by preferential exclusion, lower cold denaturation and stabilise sample
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16
Q

why are some formulations lyophilised?

A

• Many formulation are lyophilised so you need to reconstitute them. Usually lower concentrations of proteins you need to admin usually 1-10mg/mL

17
Q

Explain why the rate of ILRA degradation increased with increasing temperature and what effect this would have on the ILRA protein structure.

A
  • An increase of the physical and/or chemical degradation of the protein under heat stress is observed and may lead to instability/exposure of hydrophobic region.
  • Together with increased collision of solvent / solute molecules at higher temperatures, increased conformational change and (partial) unfolding of the protein, results in aggregation & increased turbidity (note: turbidity measures the presence of particles as they scatter more light but is also observed at high concentration: thus only the change of turbidity is relevant).
18
Q

Compare the rates of degradation and rates of increase in turbidity at the different temperatures at low sucrose concentrations and explain why degradation may have led to increased turbidity.

A

Lower rates of degradation and less turbidity observed at higher sucrose concentrations, with small/insignificant rates at 8°C and 30°C, but with a significant basal rate at 50°C unchanged by sucrose concentration.
The more compact and tightly folded protein conformation at higher sucrose concentrations may be more resistant to unfolding and side chain reactions at higher temperature.
Consequently, formation of large aggregates is less favoured at higher sucrose concentrations. This may be related to the increased chemical potential and surface area of more unfolded conformations and denatured states.

19
Q

Compare and explain why the degradation rates were lower at higher sucrose concentrations

A

Lower rates of degradation and less turbidity observed at higher sucrose concentrations, with small/insignificant rates at 8°C and 30°C, but with a significant basal rate at 50°C unchanged by sucrose concentration.
The more compact and tightly folded protein conformation at higher sucrose concentrations may be more resistant to unfolding and side chain reactions at higher temperature.
Consequently, formation of large aggregates is less favoured at higher sucrose concentrations. This may be related to the increased chemical potential and surface area of more unfolded conformations and denatured states.

20
Q

Explain how the concept of preferential exclusion may apply.

A
  • Sucrose (hydrophilic – attracts water molecules) stabilising excipient is preferentially excluded from the surface of the protein, which increases the chemical potential or energy of the unfolded state.
  • The degree of preferential exclusion and the increase in chemical potential are directly proportional to the surface area of protein exposed to solvent.
  • System will minimise thermodynamically unfavourable effects of preferential exclusion of sucrose by favouring the state with the smallest surface area / lower chemical potential / native state.
  • As the unfolded / denatured state will have a higher surface area, and increased chemical potential, with preferentially excluded sucrose, the free energy of denaturation is increased, when the native state would be favoured thermodynamically.
  • Based on this thermodynamic principle, increased sucrose should also favour the most compact protein conformation, even under non-denaturing conditions, when the protein would expected to fold more tightly.
  • A more compact/tightly folded conformation would be expected to be of smaller apparent size, when increases in sucrose concentration would be expected to result in more compact conformations and smaller apparent protein size.
21
Q

what is a denaturant?

A

interaction with polypeptide backbone of protein e.g. urea with most amino acid side chains (e.g. by H bonding)
• higher interaction when unfolded +ve concentration difference between local and bulk domain.

22
Q

what is a protectant?

A
  • lower interaction (e.g. hydrophobic) & higher exclusion when unfolded  -ve conc difference between local & bulk.
  • Preferential interaction: change in protein chemical potential in presence of co-solvent or change in local/bulk concentration to maintain constant chemical potential. Difference in preferential interaction can be related to free energy change.
23
Q

what are the long term stability testing temperatures you should use?

A

<20 +/- 5 degrees . this is room temp
5 +/- 3 degrees this is fridge temp
30 +/- 2 degrees at 65% . this is body temp

24
Q

what are the temperatures you should use for accerlerated stability testing?

A

5+/- 3 degrees
25 +/- 2 degrees at 60%
40 +/- 2 degrees at 75%

25
Q

what do you need to keep a record off when you are carrying out stability tests?

A

data must include all container closure systems

26
Q

how often should you test stability if the shelf life is under a year?

A

monthly for the first 3 months

and then 3 month intervals there after

27
Q

how often should you test stability if the shelf life is over a year?

A

every 3 months during the first year
every 6 months for the second year
annually there after