Biologics Issues and stabilisation Flashcards
For mAbs and other therapeutic proteins in solution, the hydrophobic AA should be buried in the __a__ and the hydrophilic AA should be in the __b__ .
a) Protein core
b) shell
Hydrophobic = non polar, aliphatic side groups, aromatic side chains
Hydrophillic = polar uncharged side groups, polar charged, negatively charged
Most likely to have non polar groups __A__ protein. Hydrophillic groups most likely to be on __B__.
a) inside
b) outside
What does the presence of salt bridges do to the conformation of the protein?
The presence of salt bridges “rigidifies” the conformation (can still move)
Consequences of unfolding of protein?
- Unfolding (denaturation or exposure of hydrophobic core) may lead to aggregation.
- Protein is a mix of various systems.
- If the integrity is altered, It can unfold and lead to denaturation and lead to exposure to hydrophobic core.
are mAbs colloids?
• mAbs (and other proteins) are not colloids
▫ Not a uniform distribution of the same charge (there is a variation of charges)
How to stabilise biologics?
• Need to consider attractive and repulsive forces but also hydrophobicity
▫ And consequently pH, buffer, salt (concentration and type) and concentration of co-solutes
Examples of stresses that can lead to a change in ;conformation of protein:
Changes in pH, T, p, ionic strength, concentration of co-solutes all can result in unfolding the protein thus ultimately aggregation
Increasing T or p=
Increased movement of protein and favours unfolding
Reduces reversible aggregation in favour of irreversible aggregation (something you really want to avoid).
Decreasing T ____ rate of aggregation:
Decreases
Pressure if extremely important when considering biologics as:
most of the proteins will be injected which requires pressure on syringe = sheer force which can potentially unfold protein.
Influence of pH on aggregator?
• Changing pH alters the charge of the protein and increasing ionic strength results in electrostatic forces to be felt a short distances
▫ Shifts of pH towards isoelectric point or high ionic strength tend to favour aggregation because you have a particle which isn’t charged.
Examples of chemical degradation:
Oxidation, deamidation, hydrolysis
May lead to instability then aggregation
- Exposure hydrophobic regions (will try to avoid contact with water)
- Exposure of cysteine residues of formation of disulfide bridges
Examples of physical destabilisation:
▫ Extreme pH ▫ Shear forces, (pressure you put on the plunger of syringe when you inject) ▫ Air-water interfaces ▫ Adsorption to solid surfaces ▫ Freezing drying ▫ High pH or T
What is preferential binding?
Preferential binding = co solute will bind to the surface of your protein and this will have an effect.
What is preferential exclusion?
Preferential exclusion of co-solute: co-solute mainly out of the solvation shell of the protein. Could increase the chemical potential of the unfolding protein interface.
which increases the thermodynamic stability of the native protein relative to the unfolding larger difference energy makes it less likely to unfold and lead to aggregation.
Preferential interaction example:
Denaturant: interaction with backbone of protein e.g. urea H-bonding with most AA side chains
How does sucrose work as a protectant (preferential exclusion)
Lower interaction with protein but not hydrophobic leads to higher concentration of co-solute in bulk (and will attract the water) than in the solvation shell of the protein. Makes it shrink, tighter and less likely to unfold
Stabilisation mechanism of amino acids:
Preferential hydration, preferential exclusion, decrease protein protein interactions, increase solubility, reduce viscosity
Stabilisation mechanism of polymers:
Competitive absorption, steric exclusion, preferential exclusion, preferential hydration
Stabilisation mechanism of surfactants:
Competitive absorption at interfaces, reduces denaturation at air/water interfaces
How does acylation work in terms of stabilising proteins?
Acylation with fatty acid to increase binding affinity tp serum albumin resulting in longer acting insulin, glucagon and interferon
How does PEGylation work in terms of stabilising proteins?
To reduce plasma clearance rate and achieve less frequent admin. However, some binding proteins less active when PEGylated
Stability testing for biologics is defined in the:
ICHQ5C
How is shelf life determined?
during long term stability (real time/real temperature data)
