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Medicine Pharmaceutics > Biologics 2 > Flashcards

Biologics 2 Flashcards

1
Q

in mAbs the hydrophobic amino acids are where?

A

buried in the protein core

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2
Q

in mAbs the hydrophilic AAs are?

A

the outer shell

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3
Q

charged groups on mAbs want to be where? why?

A

on the outside to interact with water

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4
Q

if the charged groups are on the inside of the mAbs what will happen?

A

they will change the confirmational structure of the protein to get to the water

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5
Q

mAbs 3d confirmation is?

A

relatively flexible

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6
Q

what can unfolding lead to?

A

denaturation and aggregation

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7
Q

non polar molecules are hydro_____

A

phobic

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8
Q

what will have the biggest impact on protein confirmation

A

the environment its exposed to

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9
Q

why does aggregation occur on denaturation

A

If the hydrophobic core gets exposed and cant refold then 2 proteins will come together to minimise contact

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10
Q

TF: mAbs and other proteins are colloids. what does this mean

A

false
theyre not
you get variation where its negative, positive or uncharged

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11
Q

what are colloids charge wise?

A

tend to be uniformly charged

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12
Q

examples of stress on a protein

A 
changes in 
pH
temperature 
ionic strength 
co solutes concentration
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13
Q

TF: the concentration of co solutes can unfold a protein

A

true

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14
Q

each AA has its own ___ and ________

A

pKa and isoelectric point

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15
Q

increasing T or P reduces _____ aggregation in favour of _______ aggregation

A

reversible

irreversible

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16
Q

decreasing T _____ the rate of aggregation

A

decreases

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17
Q

P promotes protein unfolding close to…..

A

interfaces

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18
Q

shifts in pH towards the isoelectric point (when the protein is uncharged) or high ionic strength, tends in favour of ______ ______

A

irreversible aggregation

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19
Q

why is it important to consider pressure with mAbs?

A

gets injected

pressure in syringe

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20
Q

what can happen at the surface of liquids?

A

protein may go to the interface and unfold, hydrophobic core will be in contact with the air and the rest will be in contact with the water. This can also happen on the surface of the solid. Issue with syringes which are made of silicone

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21
Q

protein unfolding at the interface is an issue with syringes made of?

A

silicone

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22
Q

ways a protein can be chemically degraded?

A

oxidation
deamination
hydrolysis

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23
Q

2 processes of aggregation?

A

exposure of hydrophobic regions to evade water contact

exposure of cys residues of formation of di-sulfide bridges

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24
Q

ways a protein can be physically degraded?

A 
pH
shear forces 
interfaces 
adsorption 
freeze drying 
high temperature
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25
Q

______ degradation is the most common type in proteins

A

physical

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26
Q

in preferential exclusion of co solute, the co solute is mainly…..

A

out of the salvation shell of the protein

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27
Q

TF: including a preferential exclusion co solute increase the chemical potential of the unfolded protein water interface more than the native one?

A

true
in the unfolded state more than the native state
therefore larger water protein interfacial surface area for the unfolded state

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28
Q

preferential binding is when the co-solute binds to the….

A

surface of the molecule

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29
Q

preferential ______ is a denaturant

A

interaction

30
Q

preferential _______ is a protectant

A

exclusion

31
Q

how can preferential binding act as a denaturant?

example?

A

cosolvent interaction with backbone of protein e.g. urea H-bonding with most AA side chains
Examples: urea or guanidine hydrochloride (works at lower conc than urea)
Unfolds the protein

32
Q

overall effect of preferential interaction as a denaturant

A

unfolds protein

33
Q

how can preferential exclusion act as a protectant

A

 Lower interaction with protein but not hydrophobic leads to higher concentration of co-solute in bulk than in the solvation shell of the protein- attract the water and render the surface of the protein less solvated- makes it tighter and less prone to unfolding- VERY SIMLIFIED EXPLANATION

34
Q

example of a protectant

A

sucrose- varies with concentration

35
Q

examples of a denaturant

A

urea

guanidine hydrochloride

36
Q

guanidine hydrochloride works at a ______ concentration than urea for a denaturant

A

lower

37
Q

how do amino acids stabilise?

A

Preferential hydration= preferential exclusion aka will be hydrated, decrease protein-protein interactions, increase solubility, reduce viscosity- good if need high concentrations
Good to use as natural in the body- safe.

38
Q

how do polymers stabilise/

A

Competitive absorption, steric exclusion, preferential exclusion, preferential hydration
Especially when you have a hydrophilic polymer e.g. PEG

39
Q

how to polyols stabilise?

A

Preferential exclusion, accumulation in hydrophobic regions.

40
Q

how do salts stabilise

A

Hoffmeister series exclusion or hydration- size and charge will effect

41
Q

how do surfactants stabilise?

A

Competitive absorption at interfaces, reduces denaturation at air/water interfaces

42
Q

explain how acylation can create a stabilising modification of therapeutic proteins

A

Acylation with fatty acid to increase binding affinity to serum albumin resulting in longer acting insulin, glucagon and interferon.
Albumin has a Fc region so gets recycled, want to link it to this.

43
Q

explain how PEGylation can create a stabilising modification of therapeutic proteins

A

To reduce plasma clearance rate (as less recognised) and achieve less frequent administration. However, some binding proteins less active when PEGylated

44
Q

modification of amino acid sequence to remove hotspot will most likely cause?

A

aggregation

45
Q

TF: stability testing biologics is the same as for other medicines

A

false

46
Q

how is shelf life determined?

A

long term stability tests

47
Q

point of accelerated studies?

A

Support to establish the shelf life- can stress them to see how they change. Can compare this to the same medicines that have been in long term storage to see if it’s the same.
Provide info on changes, validation of stability tests
Generate help to understand degradation profiles

48
Q

what are stress studies?

A

Representative accidental exposures- e.g. shaking- AZ has a robot mimicking the movement in the trucks, aeroplanes.
Reveal patterns of degradation

49
Q

long term testing temperatures ?

A 
<20 +-5
5+-3
25+-2/ 60%RH 
or
30+-2/ 65% RH
50
Q

Accelerated stability testing temperatures?

A

5 +-3
or
25+-2/60%RH

25+-2/ 60%RH

40+-2/ 74%RH

51
Q

what is the shelf life range for biologicals

A

0.5-5 years

52
Q

if the shelf life is a year or less, how often should long term stability studies be taken?

A

monthly for the first 3 month

3 month intervals thereafter

53
Q

if the shelf life is a year or more, how often should long term stability studies be taken?

A

Every 3 months during first year
Every 6 months during second year
Annually thereafter

54
Q

low temperatures _____ half life

A

extend

55
Q

why can freezing lead to damage of the biologicals?

A

cold denaturation

as a result of changes in pH, ionisation, solubility or H-bond energy

56
Q

why does repeated freezing and thawing cause aggregation?

A

pH and concentration changes

and by provision of nucleation points at ice water interfaces

57
Q

when you freeze the biologicals what freezes first?

A

the water

58
Q

what is needed if something is going to be freezes. example?

A

cyroprotection
sugars
polyhydri alcohols
AAs

59
Q

how do these cyroprotective agents work?

A

work by preferential exclusion, lower cold denaturation and stabilise sample

60
Q

what happens in nucleation of ice?

A

forms small crystals

61
Q

nucleation is below the ______ temp of water

A

fusion

62
Q

if you cool slowly you get ______ crystals

A

larger

63
Q

if you cool fast you get ______ crystals. why is this a problem?

A

small

eutectic solid formed: 2 solids- water and proteins and excipients very concentrated

64
Q

to control uniformity you should cool it at _____ temperatures

A

low (-70) worked best

65
Q

water freezes first at the ________. this means

A

interface
gets very concentrated towards the centre of the vial- this decreases as you freeze to lower temperatures as the molecules dont have as much time to move

66
Q

when you thaw what must you do?

A

swirl to get sample uniform

not shake as can damage

67
Q

lyophilised proteins have ______ term stability than liquid ones

A

longer

68
Q

why are lyophilised proteins prone to aggregation?

A

undergo reversible conformational changes during the different steps of lyophilisation with render them prone to aggregation (and similarly again when reconstituted).

69
Q

what should you do to lyophilised medicines to reduce aggregation?

A

refrigerate

hygroscopic- sealed to avoid water absorption

70
Q

what occurs in lyophilisation

A

When totally frozen in the vial you make a vacuum- all the water will instantly be taken out (drying process) as the ice sublimes from solid to gas
Process of freeze drying:
Freeze: product is completely frozen in a vial
Vaccum: product is placed under deep vacuum well below triple point of water
Dry: heat energy is added causing ice to sublime

71
Q

are there areas of high and low concentrated mAb and cosolvent in lyophilised proteins

A

yes- you get ice crystals forming first and solutes get concentrated. so highly concentrated sections of mAb and cosolvent

Medicine Pharmaceutics (6 decks)

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