Biologics 2 Flashcards
What are the 2 ways in which mAb’s can be formulated?
As liquid (low to high conc.) Lyophilised products reconstituted prior to use (usually in hospital settings)
What are mAb’s made up of?
Protein sequence of alternate charged and uncharged AA’s and hydrophobic/hydrophilic groups, this results in a flexible chain governed by attractive, repulsive and hydration forces
Is the structure of mAb’s rigid?
Not completely, it can change in response to its environment
How are the AA’s arranged (hydrophobic/hydrophilic, non-polar/polar)?
In solution, the hydrophobic AA’s should be buried in the protein core and the hydrophilic AA’s should be in the shell
Non-polar AA’s are most likely to be buried in the protein, but they can also be in the shell
Polar AA’s are less likely to be in the core and more likely to be in the shell
Changes in pH and salt content…
Will affect charges, this leading to folding or unfolding of the protein
Attractive and repulsive forces…
Create a 3-D conformation (a dynamic protein which is relatively flexible)
How does the presence of salt bridges influence structure?
Rigidifies structure
How do mAb’s differ to colloids in their structure (charge)?
A 3-D conformation will likely have positive, negative and uncharged residues (hydrophilic and hydrophobic) in its structure
Whereas, a colloid can only have one charge, positive or negative at a time (uniform distribution of one charge)
What is a colloid?
Homogenous substance containing particles of one substance dispersed throughout another
Can DVLO theory be used to describe the stability of protein solutions?
This is indicative at best due to the differences between proteins and colloids
What extra factors need to be considered when thinking about the stability of proteins?
Attractive and repulsive forces, hydrophobicity, buffer, pH, salt (conc. and type), conc. of co-solutes
Changes in pH, temp, pressure, ionic strength, conc. of solutes result in what?
Partial or complete unfolding of the protein thus aggregation and therefore loss of therapeutic use of immune response
What is the effect of increasing temperature or pressure on a mAb?
Increasing temperature reduces reversible aggregation (agglomeration) in favour of irreversible aggregation
What is the effect of decreasing temperature on a mAb?
Decreasing temperature slows the diffusion of molecules, conformation changes are reduced, thus less likely to expose hydrophobic pockets, so decreases rates of aggregation
What happens when two hydrophobic pockets become exposed?
They will bind in order to limit their exposure to the aqueous environment
How are proteins usually administered and what effect does this have on associated pressure-related instability?
Proteins are administered via syringes or infusion, go from a large diameter in the container to a small diameter in the needle, thus resulting in a pressure increase
Proteins will be in non-Newtonian fluid, which on the walls of the capillaries or syringe will be likely to unfold, leading to aggregation
What is the effect of changing pH on a mAb?
Changing pH will alter the charge of the protein and increasing ionic strength results in electrostatic forces to be felt at short distances
What is the effect of a change of pH (moving closer to pI)?
Will result in a decrease of the overall positive charge, resulting in a less repulsive particle, relying on the polar AA’s for its solvation and increasing the likelihood of hydrophobic AA exposure (partial unfolding) and aggregation
What is observed at high ionic strength?
When high concentrations of salt, e.g. NaCl, are added, the repulsive forces are screened (damping of electric fields caused by presence of mobile charge carriers) and the attractive forces become prevalent, leading to aggregation
Aggregation occurs in mAbs, but where else does aggregation occur?
What is valid for mAb’s is also valid for disease or in life as aggregation is the process that leads to the formation of collagen fibres and to the fibres observed in Alzheimers and ocular disease
The only difference for mAb’s is that the aggregation is amorphous, whilst for others, organised fibres are observed
What is the key aspect needed for aggregation?
Partial or complete unfolding
Where does unfolding occur?
When the mAb is in contact with an interface (water/container surface, water/silicone, air/water)
Air/water is the main one
Why does unfolding occur?
Unfolding at the interface allows hydrophobic patches to evade contact with water
These unfolded proteins may then regroup and form aggregates
What are the 3 mechanisms of chemical degradation and what do they lead to?
Oxidation, deamidation and hydrolysis
May lead to instability and then aggregation by exposure of hydrophobic regions, exposure of Cys residues or formation of disulphide bridges
What are the 6 mechanisms of physical destabilisation?
Extreme pH
Shear forces (e.g. plunger depression)
Air-water interfaces (e.g. head collar in syringe)
Adsorption to solid surfaces
Freeze drying
High pH or temperature (e.g. close to pI, no charge, poor solubility)
