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
How are surfactants used as stabilisers?
Competitive absorption at interfaces, reduces denaturation at air/water interfaces
How is acylation used in stabilising modification of therapeutic proteins?
Acylation with fatty acid to increase binding affinity to serum albumin (has an Fc region - can be recycled) resulting in longer acting insulin, glucagon and interferon
How is PEGylation used in stabilising modification of therapeutic proteins?
To reduce plasma clearance rate and achieve less frequent administration. However, some binding proteins less active when PEGylated
How is surface engineering used in stabilising modification of therapeutic proteins?
Modification of AA sequence to remove hotspot likely to cause aggregation
How is shelf life determined?
Determined during long term stability testing (real time/real temperature data)
What are accelerated stability testing studies?
o Support to establish the shelf life
o Provide info on changes, validation of stability tests
o Generate help to understand degradation profiles
o Test conditions are normally done earlier than real storage conditions
o Can be done on new products to see what happens in long term
What are stress studies?
o Representative accidental exposures – e.g. shaking during transportation
o Reveal patterns of degradation
What conditions are used for long term stability testing?
- storage at room temp
What conditions are used for accelerated stability testing?
+5° ± 3°C and /or
+25° ± 2°C/60%RH
+40° ± 2°C/75%RH
Why can’t you go above 40 degrees in stability testing?
Protein tends to unfold between 40 and 58 degrees - as soon as protein unfolds, mechanisms are created that cannot be controlled
What are used in stress testing?
Temperature, pH, light, oxidation, shaking, freeze thaw
What must all data provided be?
With container closure systems used
What is the guidance for long term testing for shelf life for biologics?
0.5-5 years shelf life
If you are looking for a shelf life less than a year, what is the testing frequency?
Monthly testing for the first 3 months and then 3 month intervals thereafter. Testing points (months): 0,1,2,3,6,9 and 12
If you are looking for a shelf life MORE than a year, what is the testing frequency?
Every 3 months during the first year and every 6 months during the second year and annually thereafter. Testing points (months): 0,3,6,9,12,18,24,36,48...
What techniques are used for stability testing?
HPLC, Mass spec, UV, light spectroscopy, IEF (isoelectric focusing)
What affect does low temperature have on shelf life and what problems can it lead to?
Extends shelf life BUT cold denaturation which happens when freezing sample may lead to damage as freezing results in change of pH, ionisation, solubility or H-bond energies
What can repeated freezing and thawing cause?
Aggregation by pH and concentration changes and by provision of nucleation points at ice water interface. Every time you freeze, water freezes first and this increases the concentration in ions close to the protein
What can be used to lower cold denaturation and stabilise the sample?
Cryoprotection by sugars, polyhydric alcohols, AAs, work by preferential exclusion to lower cold denaturation
What happens to crystals when you cool slowly?
Larger crystals
What happens to crystals if you cool quickly?
Small crystals - may cause issues
Why is there much more protein when you take samples from the centre of the vial as opposed to the side of the vial?
Cold comes from the outside - water molecules on the outside freeze more quickly, making it more concentrated in protein and salt. This is why concentration is higher inside
What can be done to control uniformity of concentrations?
Cool to low concentrations/ temperatures
What stability do lyophilised protein formulations have?
Greater long-term stability
What changes do lyophilised proteins undergo?
These do undergo reversible conformational changes (PRONE TO AGGREGATION = BAD) during the different steps of lyophilisation with render them prone to aggregation (and similarly again when reconstituted).
What concentrations of proteins is protein lyophilisation used?
Low concentrations: 1-10mg/ml
What reduces aggregation rates in lyophilised medicines?
Storing them in fridges
Are lyophilised proteins hygroscopic?
YES - sealed to avoid water vapour absorption
What happens during freezing (lyophilisation)?
• During freezing (lyophilisation) ice crystals form first and solutes get concentrated.
What is the process of freeze drying?
o Freeze: product is completely frozen in a vial
o Vacuum: product is placed under deep vacuum well below triple point of water
o Dry: heat energy is added causing ice to sublime
What is the double funnel model?
The double funnel model describes the conformational space of selection of folding or aggregation from intermediate states, whose architecture depends on environmental conditions. Aggregation funnel is less jagged than the native funnel, due to lower restrictions on aggregate conformations.
How do low temperatures extend shelf life?
As temperature drops, properties of solvent change including dielectric constant, acid/base ionization, diffusion rates, solubility of hydrophobic residues and hydrogen bond energies
How does cryoprotection by sugars, polyhydric alcohol, oligosaccharides, amino acids and surfactants work?
• Largely work by preferential exclusion, lowering cold denaturation temperature and stabilising osmotic stresses, whereas surfactants interfere with ice / water interface
What can be done with frozen vials to avoid mis-dosing?
Gently mix frozen vials to avoid mis-dosing
*Concentration gradients are formed during freezing, and tend to remain, if thawing performed without mixing. Vial shape, size & materials affect freezing pattern.
What are used for sustained release biologics?
Polymer implants e.g. contraceptives, growth hormones
