protein stability Flashcards
what are the major adv of Protein Pharmaceuticals ?
- High specificity and strong potency
- Relatively low concentrations so less side effects
what are the major challenges of Protein Pharmaceuticals ?
antigenicity
stability
Injection so less convenient delivery
what can affect protein antigenicity?
- Pharmaceutical proteins are foreign as produced from non-human host cells like E. coli and Chinese hamster ovary CHO cells
- If downstream processes not conducted properly —> Impurities —> Immunogenic response (mild allergic reaction vs anaphylactic shock)
- Long term injection of same protein —> Patient may develop antibodies against exogenous protein —> Protein attacked —> Protein loss efficacy over time —> So can increase dosing but also means more side effects
how can a protein loss it’s biological activity?
through loss of 3d conformation via:
- Denaturation
- Covalently modifying protein
- Partially degrading protein
when can the loss of protein’s biological activity occur?
- Protein recovery phase
- Protein purification process
- Post-protein purification (storage)
- Bacterial contamination —> Produce proteases that conduct slow proteolysis on proteins
- Storage in solution: Water is weakly nucleophilic so can cause slow hydrolysis of protein —> protein degradation —> So store in freeze-dried form
- Solution (shelf life few days-1 month) VS Freeze-dried form (shelf life 2-3y)
how is the potency of the protein affected?
- Difficult to change purity of protein because protein stays in unit dose
- On storage over 12 months, proteins start to unfold slightly —> Potency affected
- Conduct biological assays to see if stability will affect activity and potency of protein, physical and chemical assays are inadequate
What are the mechanisms that lead to instability of proteins?
- Physical: Protein Aggregation
- Chemical
what are the stages where protein aggregation can occur?
- N stage: Protein folded in optimal 3D conformation (native conformation)
- U stage (reversible by restoring back favourable conditions): When unfavourable conditions are introduced (eg. heat) —> Protein start to unfold to reach U stage, protein still active but start to ppt out
- A stage (irreversible): If continue to subject U stage to unfavourable conditions —> protein continue to unfold till aggregated stage, have ppt out, protein lost all or most of activity —> Immunogenicity
why do proteins aggregate?
Due to intermolecular association of partially denatured protein chains —> Fully unfolded polypeptide chains tend to aggregate tgt due to hydrophobic interactions
what are the Factors inducing aggregation?
- temperature
- Increase temp —> disrupt non-covalent forces —> promotes protein unfolding —> denatured protein aggregation —> irreverisble denaturation
- pH
- extreme pHs —> change in ionisation status of side chains of amino acid residues —> disrupt conformation —> protein unfold and aggregate
- Can contribute to chemical instability
- Hydrolysis of Asp residues
- Deamidation of Asn and Gln
- Ionic strength
- Adsorption (stuck)
- Proteins can be adsorbed to many surfaces (esp plastic) and interfaces —> Change in secondary or tertiary structure —> Destabilisation of protein —> Protein aggregation
- Use glass bottles
- Shaking and shearing
- Agitation —> Air introduced —> Surface of air bubbles creates an air-liquid interface that trap proteins —> Change in conformation of proteins —> Unfolding of proteins to expose hydrophobic core —> Protein aggregation
- Addition of Non-aqueous solvents
- Organic solvents: Ethanol, Propanol —> Decrease polarity of aqueous solvent containing proteins —> Decrease ability of formation of hydrogen bonds between protein and water —> Disrupt hydration shell that surrounds and hence stabilises proteins —> expose hydrophobic core —> Protein aggregation
- Repeated freeze-thaw
- Freeze —> Sharp ice crystals form and pierce through 3D conformation of protein —> decrease stability of protein —> Unfold —> and thaw and freeze and thaw —> further unfolding —> protein aggregation
- Photodegradation (exposure to light) of amino acids
- Tryptophan very susceptible, where its side chain can absorb UV rays and undergo changes
- Store proteins in amber bottle for increase stability of proteins
- Vortexing
- Chemical modification of proteins —> Expose hydrophobic sites to aggregate by hydrophobic interactions
what other factors can cause protein aggregation?
Simultaneous chemical and physical factors can cause protein aggregation
why can chemicals cause protein aggregation?
Proteins consist of amino acids —> reactivity of side chains of amino acids —> susceptible to chemical reactions
note: more than 1 reaction can happen simultaneously
what determines extent of chemical reactivity of protein aggregation?
- Localisation of susceptible amino acid can determine extent of chemical reactivity
- For same AA that is located on protein surface, it is usually more exposed to chemical reactions as compared if located in core of protein
- Chemical reaction does not always mean loss of conformation or activity —> need to test to determine extent
what are the Types of Chemical reactions that cause protein aggregation?
- Deamidation
- More susceptible amino acids: Asn and Gln
- Rmb that location matters!
- Oxidation
- Can occur to side chains of His (H), Met (M), Cys (C), Trp (W), Tyr (Y)
- Presence of transitional metal ions —> Closer proximity to metal binding sites generates reactive oxygen species —> Increase oxidation rates
- Most susceptible amino acids: Tyr and Cys
- Proteolysis
- Disulfide bond breakage and formation
- Type of oxidation that only happens to Cys due to S groups between two Cys molecules can join together to form disulfide bond
- Presence of disulfide bond —> good or bad is determined through testing
- Good: Favourable for protein conformation or activity
- Bad: Unfavourable for protein conformation (if native form required) or activity (if reduced form required)
- Hydrolysis of peptide bonds
- Especially under acidic or basic pH
- More susceptible amino acids: Asp-Gly and Asp-Pro
what are the Ways to stabilise final liquid formulations?
- Substitution or chemical modification of protein
- Add different agents into final formulation
how to do Substitution or chemical modification of protein to increase its stability?
note: wont affect overall activity
- Modify or Substitute susceptible AA with another AA
- Via site-directed mutagenesis
- Eg. Cys replaced by Ser
- Introduce disulfide bond to stabilise folded form
- May not work as may be unfavourable to protein (determined by testing)
- PEGlyation (very common)
- Conjugating polyethylene glycol (PEG) to a protein —> Keep in native form longer, Increase circulation time in blood
- Eg. Recombinant proteins conjugated with PEG —> PEGlyated proteins
- Acylation
- Conjugating lipophilic fatty acids to protein —> makes overall complex more lipophilic so expel water —> Upon storage, protein scared of water because water is weak nucleophile —> maintain stability
what kind of agents to add into final formulation to increase protein stability?
- Stabilisers (sugar, polyols)
- Solubility enhances (Lysine, arginine, surfactants)
- Anti-adsorption and anti-aggregation agents (albumin, surfactants)
- Buffer components (phosphate salts): Prevent very acidic or alkaline conditions
- Preservatives and anti-oxidants (inert gas, thimerosal, phenol, benzyl alcohol)
What is recombinant proteins?
- Artificial/ Synthetic protein synthesised by host cell, not by own body
- Immunogenicity issues —> Shift from animal to recombinant proteins
- FDA: Manufacturing steps should avoid animal products, Animal component-free media to be used in host cell culture
