Biologics: issues with stabilisation and formulation Flashcards
What influence do mAbs have on conformation on stability?
- Proteins are made from amino acids - divided into hydrophobic and hydrophillic
- Most likely to have non-polar groups buried inside the protein – evade contact with water – decreases when you have polar and charged groups
- Least charged proteins (amino acids) will be outside the structure – want to be in contact with water – putting them inside would change equilibrium
What conformation is seen in mAbs?
- 3D conformation: dynamic, protein is relatively flexible.
- has salt bridges which rigidify the conformation
- unfolding can occur
What can unfolding in mAbs lead to?
May lead to aggregation and will try to evade the contact of water
Do mAbs have uniform distribution of the same charge?
No - need to consider attractive and repulsive forces but also hydrophobicity
What can result in unfolding the protein thus leading to aggregation?
Changes in pH, Temperature, pressure, ionic strength, concentration of co-solutes (some will attract water – so favour condensation and therefore stabilising)
What reduces reversible aggregation?
Increasing temperature or pressure (want to AVOID this)
What does decreasing Temperature do?
Decreases rate of aggregation
What does pressure promote regarding proteins?
Pressure promotes protein unfolding close to interfaces - they are injected, which requires pressure (mAbs)
What pH changes favour aggregation?
Shifts of pH towards isoelectric point (uncharged) or high ionic strength tend to favour aggregation
What is aggregation valid for?
Valid for mAbs solution, aggregation exists in nature or disease (collagen, Alzheimer, ocular disease)
What might happen if a syringe is coated in silicone?
May react with protein
What is chemical degradation in protein instability?
o Oxidation, deamidation, hydrolysis
o May lead to instability then aggregation
- Exposure hydrophobic regions
- Exposure of cysteine residues of formation of disulfide bridges
What is included in physical destabilisation of proteins?
o Extreme pH
o Shear forces – pressure you put on the plunger of the syringe
o Air-water interfaces – adsorption to interface
o Adsorption to solid surfaces
o Freezing drying
o High pH or T
What is preferential binding?
Co-solute molecules are within the solvation shell of the protein.
Co-solute will bind to the surface of molecule
What is preferential exclusion?
Co-solute molecules are outside the solvation shell of the protein. Excipients are excluded due to larger protein-water interfacial surface area thus adding preferentially excluded co-solvent could increase the chemical potential of the unfolding water interface more than the negative one.
What does preferential exclusion increase?
Increases the thermodynamic stability of the native protein relative to the unfolding
What does a larger difference in energy in preferential exclusion mean?
Makes it LESS likely to unfold and lead to aggregation
What is preferential binding/interaction called and examples?
Denaturant.
- co-solute will interact with the backbone of protein e.g. Urea H-bonding with most AA side chains
- Binds to surface of protein and unfolds the protein
- e.g. Urea or guanidine Hcl
What is preferential exclusion called and examples?
Protectant
- lower interaction with protein but not hydrophobic leads to higher concentration of co-solute in bulk (ATTRACTS WATER - makes it shrink and less subject to unfolding) than in the solvation shell of the protein
- e.g. sucrose
How are amino acids used as a stabiliser?
Natural, safe compound present in the body: preferential hydration, preferential exclusion, decrease protein-protein interactions, increase solubility, reduce viscosity (arginine)
How are polymers used as stabilisiers?
Competitive absorption, steric exclusion (stop contact), preferential exclusion, preferential hydration
How are polyols used as stabilisers?
Preferential exclusion, accumulation in hydrophobic regions
How are salts used as stabilisers?
Hoffmeister series exclusions or hydration
