Biologics II Flashcards
What types of amino acids are considered hydrophobic?
- non-polar, aliphatic side groups
- aromatic side groups
What types of amino acids are considered hydrophilic?
- polar, uncharged side groups
- positively charged side groups
- negatively charged side groups
What is the fraction highly buried?
- the amount of protein that is buried in the core, to limit contact with water
- fraction highly buried of hydrophobic AAs is large whilst hydrophilic is small
What rigidifies the otherwise flexible protein structure?
salt bridges
How does the unfolding (denaturation) of the protein lead to aggregation?
- hydrophobic core is exposed to hydrophilic environment
- to limit contact, core then binds to the exposed core of another protein molecule
mAbs and other proteins are colloids, true or false?
FALSE because they do not have a uniform distribution of the same charge
made up of different AAs, where you will have different patches of different charges
What needs to be factors need to be considered about a proteins stabilisation?
- attractive and repulsive forces
- hydrophobicity of the environment
What stresses induce changes of the conformation of a protein?
- changes in pH (each AA has their own pKa)
- changes in temperature (increasing = denaturation)
- changes in pressure
- changes in ionic strength (amount of salt in solution)
- changes in concentration of co-solutes (some solutes will attract the water and favour condensation of the protein)
different stresses are induced on the protein throughout various stages of the manufacturing process
What are the effects of increasing temperature or pressure on reversible aggregation?
- it will REDUCE the amount of REVERSIBLE aggregation
BUT
will increase the amount of irreversible aggregation, meaning that the proteins are then not recoverable
Why is it important to consider pressure with protein formulations?
- changing pressure promotes unfolding of proteins at interfaces
- important because most mAbs will be injected into a patient
- using a syringe and applying pressure when administering may potentially unfold the protein
What is turbidity?
- turbidity of the solution is where you have protein aggregates that are large enough to be able to see
How does interfaces affect protein aggregation?
- film of protein molecules that line the interface (e.g. solution/air in a container)
- shedding of the protein film (aggregate) into the bulk
What are the possible routes of chemical degradation of proteins and what could this lead to?
- oxidation
- deamination
- hydrolysis
e. g. exposure of the hydrophobic regions which evade contact with water by aggregation
e. g. exposure of Cys residues with the formation of disulphide bridges
What are the possible routes of physical degradation of proteins?
- extreme pH
- shear forces
- air-water interfaces
- adsorption to solid surfaces
- freezing drying
- high pH or temperature
How does changing the pH affect the aggregation of proteins?
- changing the pH towards the isoelectric point changes the charge of the protein
- proteins will then be uncharged
- this favours aggregation
What is preferential exclusion?
- where the co-solute excipients are outside the solvation shell of the protein
- protectant effect
- folded protein
- increases thermodynamic stability
- increases the energy
What is preferential binding/interaction?
- where the co-solute excipients are inside the solvation shell of the protein
- denaturant effect
- unfolded protein
Preferentially excluded excipients are excluded more from native or unfolded states?
- UNFOLDED
due to the larger water-protein interfacial surface area
degree of preferential exclusion and increase in chemical potential is directly proportional to the surface area of the surface area of the protein exposed to the solvent
What is the denaturant effect of preferentially binding excipients?
- within the solvation shell of the protein
- interacts with the backbone of the protein
- unfolds the protein
What is the protective effect of preferentially excluded excipients?
- outside of the solvation shell of the protein
- draws water molecules away from the protein into the bulk
- causes the protein to shrink on itself
- makes the protein more compact and less likely to unfold
- increases chemical potential
Example of a preferentially binding excipient?
- urea that will form H bonds with most AAs
- guanidine hydrochloride
Example of a preferentially excluded excipient?
- sucrose
What are the various stabilisers (excipients) of proteins?
- amino acids
- polymers
- polyols
- salts
- surfactants
How do amino acids stabilise proteins?
- preferential hydration
- preferential exclusion
- decrease protein-protein interactions
- increases solubility
- decreases viscosity
safe excipient as they are naturally found in the body
How do polymers stabilise proteins?
- competitive absorption
- steric exclusion - proteins cannot come into contact with one another to aggregate
- preferential exclusion
- preferential hydration
How do polyols stabilise proteins?
- preferential exclusion
- accumulation at hydrophobic regions
How do surfactants stabilise proteins?
- competitive absorption at interfaces
- reduces denaturation at air/water interfaces
ensures that the protein remains in the bulk where it is more likely to remain in solution and not aggregate (and doesn’t adsorb to the container)
What are the stabilising modifications of therapeutic proteins?
- acylation
- PEGylation
- surface engineering
Acylation of Therapeutic Proteins
- acylation with a fatty acid
- increases binding affinity to albumin
- albumin is recycled via the FcRn pathway
- longer acting protein molecule (insulin/glucagon/interferon)
PEGylation of Therapeutic Proteins
- reduce plasma clearance rate
- less recognised by the body to be cleared
- BUT, some binding proteins less active when PEGylated
Surface Engineering Therapeutic Proteins
- removing the hotspot that is most likely to cause aggregation of a protein
Stability testing for proteins is the same as small drug molecules, true or false?
FALSE
- defined in the ICHQ5C
What are the three different stability tests of proteins?
- Shelf Life
- Accelerated Studies
- Stress Studies
all must provide data on the container of the products used (e.g. vial + plastic top)
What are the shelf life (long term) testing conditions?
is determined using long term stability (real time/real temperature data)
- below 20 degrees +/- 5 degrees (room temp)
- above 5 degrees +/- 3 degrees (fridge)
- above 25 degrees +/- 2 degrees with 60% RH OR above 30 degrees +/- 2 degrees with 65% RH
What are the accelerated testing conditions?
supports to establish shelf life
normal done before real storage conditions
helps to understand degradation profiles
- above 5 degrees +/- 3 degrees
- above 25 degrees +/- 2 degrees with 60% RH
- above 40 degrees +/- 2 degrees with 75% RH
What are the stress studies conducted on proteins?
- temperature
- pH
- light
- oxidation
- shaking
- freeze thaw
At what temperatures to proteins begin to unfold and why is this important?
between 42 - 58 degrees
- accelerated studies should not go above 40 degrees
- stress studies may use temperatures of 40 - 45 degrees
What are the testing intervals for long term testing?
- guidance is 0.5 - 5 years shelf life for most biologics
less than 1 year testing
- monthly for the first 3 months
- 3 month intervals after
- 0, 1, 2, 3, 6, 9, 12
more than a year testing
- every 3 months for the first year
- every 6 months for the second year
- annually after that
- 0, 3, 6, 9, 12, 18, 24, 36, 48
Low temperatures can extend the shelf life of proteins, true or false?
TRUE
- but freezing leads to a different type of denaturation
Why does freezing lead to denaturation?
because it causes changes in
- pH
- ionisation
- solubility
- H bond energies
Repeated freezing and thawing causes aggregation and denaturation by which mechanisms?
- pH and concentration changes
- provision of nucleation points at ice water interface
What is a cryoprotective excipients?
an excipients that protects the protein from damage during freezing, work by
- preferential exclusion
- lower cold denaturation
- stabilise sample
What are examples of cryoprotective excipients?
- sugars
- polyhedric alcohols
- AA
What are the stages of freezing, involving nucleation of ice crystals?
- supercooling
- nucleation
- freezing point depression to Tf (changing the freeze point of the solvent to be lower by adding solutes e.g. water and salt)
- freeze-concentration
which gives you a system of two solids (one ice and one solid with all the proteins and excipients)
Nucleation
of small ice crystals in the solution, these then form ice crystals
the unfrozen fraction contains all of the proteins, salts and excipients
Freeze-Concentration
- freezing the unfrozen fraction
slowly cooling - forms large crystals of ice
fast cooling - forms lots of small crystals of ice
Where is the largest concentration of protein found in a vial following freezing and what is the importance of this?
in the centre
- water molecules on the outside of the vial will be the first affected by cold temperatures
- concentration of the protein rises (not as much water available as it is frozen)
results in
- dosing issues
- poor uniformity of content
- mixing required to get a homogenous solution
Lyophilised proteins have greater long-term stability than solutions, but for what concentration upon reconstitution?
1 - 10mg/mL
Lyophilisation means that there is no aggregation or denaturation of proteins, true or false?
FALSE
- during lyophilisation (and then again on reconstitution), undergoes reversible conformational changes that make them prone to aggregation
- reactions and denaturation continue when lyophilised
What storage conditions are appropriate for lyophilised proteins to reduce aggregation?
- refrigerate
- hygroscopic - sealed to avoid water vapour absorption
What are the stages of freeze drying?
- freeze (completely in the vial)
- vacuum (below the triple point of water so that any water is removed, ice goes straight from a solid to a gas)
- drying (heat energy to cause the ice to sublime)
