biopharmaceuticals Flashcards
process of aggregation
native <-> unfolded -> aggregated
- native: optimal 3D
- unfolded: native + unfavourable condition -> unfolded w retained activity
- aggregated: irreversible unfolding
mechanisms of aggregation
- hydrophobic force
- factors that induce aggregation
1) temp
2) pH
3) ionic strength
4) vortexing
5) chemical modification of protein
broad factors that affect protein stability
1) temp
2) pH
3) adsorption
4) shaking & shearing (agitatino)
5) non-aq solvent
6) repeated freeze-thaw
7) photodegradation
how does adsorption affect protein stability
- change secondary & tertiary structure = lose ideal conformation = destabilise
- store in glass bottle cuz lesser adherence
- adhere -> lose proteins
how does shaking & shearing (Agitation) affect protein stability
- agitation -> introduce air into protein solution -> air/liquid interface
- protein trapped in air/liquid interface -> unfold -> further expose hydrophobic residues to air -> partial/complete protein denaturation
- shearing -> expose hydrophobic areas
how does repeated freeze-thaw affect protein stability
sharp ice crystals form & pierce 3D conformation of protein when freeze -> bad for stability -> unfold
how does photodegradation affect protein stability
- risk of protein aggregation upon exposure to light
- tryptophan: side chain cleavage of Trp
- store in amber bottle
types of chemical reaction that affects protein stability
1) deamidation
- most common
- Asn, Gln
- relative position determine relative rate (more susceptible at surface)
2) oxidation
- side chain of His (H), Met (M), Cys (C), Trp (W), Tyr (Y)
- catalysed by transitional ions (create ros = higher rate)
- thiol group of C & M most easily oxidised
- depends on position
3) disulfide bone breakage & formation
- oxidation
- Cys susceptible
- either good or bad for protein stability
4) hydrolysis
- esp under acidic/basic condition
- Asp - Gly & Asp - Pro more susceptible
methods to stabilise liquid formulation
1) amino acid substitution/modification via site-directed mutagenesis
- form protein analogue
- replace deamidated Asn sites
2) introduce disulfide bond -> stabilise protein
3) PEGylation
4) acylation
- chemical attachment of fatty acid to residue on protein surface
- increase circulation time in blood
impact of recombinant protein technology on pharmaceutical product
1) overcome limited resource availability
2) production of safer biopharmaceuticals
- X blood-borne pathogens
3) provide alternative way to obtain protein-based product rather than directly isolated from infected sources
4) design desirable mutations
5) cheaper, safer, abundant supply
advantages of using insect cells for recombinant protein making
multiply faster, require lesser materials
advantages of E coli for recombinant protein making
- well characterised, good for genetic manipulation
- high expression levels of recombinant protein
- grow rapidly on simple & inexpensive media
disadvantages of E coli for recombinant protein making
- recombinant protein accumulate intracellularly -> form inclusion bodies (insoluble aggregate)
- lack ability to perform post-translational modifications
- presence of LPS on surfaces act as pyrogens
why is Chinese Hamster Ovary (CHO) cells preferred?
1) adapt & grow in suspension culture -> good for large scale culture
2) lesser risk
3) grow in serum-free media
4) allow post-translational mod to recombinant proteins
5) genetically mutate to increase yield
how to determine which cell type to use for recombinant protein making
1) size
- large proteins -> CHO
- small proteins - E. coli
2) characteristics
- post translational mod -> CHO
- require solubility & native folding -> CHO
- high yield low cost -> E.coli
