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Flashcards in Biologics II Deck (51)
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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
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?

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