Biologics 2 Flashcards
in mAbs the hydrophobic amino acids are where?
buried in the protein core
in mAbs the hydrophilic AAs are?
the outer shell
charged groups on mAbs want to be where? why?
on the outside to interact with water
if the charged groups are on the inside of the mAbs what will happen?
they will change the confirmational structure of the protein to get to the water
mAbs 3d confirmation is?
relatively flexible
what can unfolding lead to?
denaturation and aggregation
non polar molecules are hydro_____
phobic
what will have the biggest impact on protein confirmation
the environment its exposed to
why does aggregation occur on denaturation
If the hydrophobic core gets exposed and cant refold then 2 proteins will come together to minimise contact
TF: mAbs and other proteins are colloids. what does this mean
false
theyre not
you get variation where its negative, positive or uncharged
what are colloids charge wise?
tend to be uniformly charged
examples of stress on a protein
changes in pH temperature ionic strength co solutes concentration
TF: the concentration of co solutes can unfold a protein
true
each AA has its own ___ and ________
pKa and isoelectric point
increasing T or P reduces _____ aggregation in favour of _______ aggregation
reversible
irreversible
decreasing T _____ the rate of aggregation
decreases
P promotes protein unfolding close to…..
interfaces
shifts in pH towards the isoelectric point (when the protein is uncharged) or high ionic strength, tends in favour of ______ ______
irreversible aggregation
why is it important to consider pressure with mAbs?
gets injected
pressure in syringe
what can happen at the surface of liquids?
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
protein unfolding at the interface is an issue with syringes made of?
silicone
ways a protein can be chemically degraded?
oxidation
deamination
hydrolysis
2 processes of aggregation?
exposure of hydrophobic regions to evade water contact
exposure of cys residues of formation of di-sulfide bridges
ways a protein can be physically degraded?
pH shear forces interfaces adsorption freeze drying high temperature
______ degradation is the most common type in proteins
physical
in preferential exclusion of co solute, the co solute is mainly…..
out of the salvation shell of the protein
TF: including a preferential exclusion co solute increase the chemical potential of the unfolded protein water interface more than the native one?
true
in the unfolded state more than the native state
therefore larger water protein interfacial surface area for the unfolded state
preferential binding is when the co-solute binds to the….
surface of the molecule
preferential ______ is a denaturant
interaction
preferential _______ is a protectant
exclusion
how can preferential binding act as a denaturant?
example?
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
overall effect of preferential interaction as a denaturant
unfolds protein
how can preferential exclusion act as 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- attract the water and render the surface of the protein less solvated- makes it tighter and less prone to unfolding- VERY SIMLIFIED EXPLANATION
example of a protectant
sucrose- varies with concentration
examples of a denaturant
urea
guanidine hydrochloride
guanidine hydrochloride works at a ______ concentration than urea for a denaturant
lower
how do amino acids stabilise?
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.
how do polymers stabilise/
Competitive absorption, steric exclusion, preferential exclusion, preferential hydration
Especially when you have a hydrophilic polymer e.g. PEG
how to polyols stabilise?
Preferential exclusion, accumulation in hydrophobic regions.
how do salts stabilise
Hoffmeister series exclusion or hydration- size and charge will effect
how do surfactants stabilise?
Competitive absorption at interfaces, reduces denaturation at air/water interfaces
explain how acylation can create a stabilising modification of therapeutic proteins
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.
explain how PEGylation can create a stabilising modification of therapeutic proteins
To reduce plasma clearance rate (as less recognised) and achieve less frequent administration. However, some binding proteins less active when PEGylated
modification of amino acid sequence to remove hotspot will most likely cause?
aggregation
TF: stability testing biologics is the same as for other medicines
false
how is shelf life determined?
long term stability tests
point of accelerated studies?
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
what are stress studies?
Representative accidental exposures- e.g. shaking- AZ has a robot mimicking the movement in the trucks, aeroplanes.
Reveal patterns of degradation
long term testing temperatures ?
<20 +-5 5+-3 25+-2/ 60%RH or 30+-2/ 65% RH
Accelerated stability testing temperatures?
5 +-3
or
25+-2/60%RH
25+-2/ 60%RH
40+-2/ 74%RH
what is the shelf life range for biologicals
0.5-5 years
if the shelf life is a year or less, how often should long term stability studies be taken?
monthly for the first 3 month
3 month intervals thereafter
if the shelf life is a year or more, how often should long term stability studies be taken?
Every 3 months during first year
Every 6 months during second year
Annually thereafter
low temperatures _____ half life
extend
why can freezing lead to damage of the biologicals?
cold denaturation
as a result of changes in pH, ionisation, solubility or H-bond energy
why does repeated freezing and thawing cause aggregation?
pH and concentration changes
and by provision of nucleation points at ice water interfaces
when you freeze the biologicals what freezes first?
the water
what is needed if something is going to be freezes. example?
cyroprotection
sugars
polyhydri alcohols
AAs
how do these cyroprotective agents work?
work by preferential exclusion, lower cold denaturation and stabilise sample
what happens in nucleation of ice?
forms small crystals
nucleation is below the ______ temp of water
fusion
if you cool slowly you get ______ crystals
larger
if you cool fast you get ______ crystals. why is this a problem?
small
eutectic solid formed: 2 solids- water and proteins and excipients very concentrated
to control uniformity you should cool it at _____ temperatures
low (-70) worked best
water freezes first at the ________. this means
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
when you thaw what must you do?
swirl to get sample uniform
not shake as can damage
lyophilised proteins have ______ term stability than liquid ones
longer
why are lyophilised proteins prone to aggregation?
undergo reversible conformational changes during the different steps of lyophilisation with render them prone to aggregation (and similarly again when reconstituted).
what should you do to lyophilised medicines to reduce aggregation?
refrigerate
hygroscopic- sealed to avoid water absorption
what occurs in lyophilisation
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
are there areas of high and low concentrated mAb and cosolvent in lyophilised proteins
yes- you get ice crystals forming first and solutes get concentrated. so highly concentrated sections of mAb and cosolvent
