Peptide and Protein Pharmaceuticals Flashcards
1
Q
Recombinant DNA methods
A
Large scale production of specifically-modified proteins
- replace lone cysteine a.a with structurally similar serine but without the availability to produce disulphide bond
Eg Human b-interferon has 2 S-S bond from 2 cysteines which stabilises the protein but also has a third cysteine that can displace the S-S bond and destabilise the protein. Hence replace with a serine
2
Q
Preformulation of development of peptides and proteins
- background equilibrium properties of protein and peptides
A
- Hydrophobic character
- Polar character
- Solubility - lipid media
- Isoelectric point
- Adsorption
3
Q
- Hydrophobic character
A
- Protein with amino acids that have predominantly hydrocarbon side chains are hydrophobic
- Can be quantified and may be localised to specific region
- Hydrophobicity can impact on solubility and surface adsorption
4
Q
- Polar character
A
- amino acids with weakly ionisable side chains
- Diff pKa depending on surroundings/ microenvironment
- Groups are weaker when microenvironment is largely hydrophobic (due to dependence on water to remove/ donate protons). Hydrophobic groups immobilise water molecules by forming hydrogen-bonded water sheaths around them –> reduce proton mobility –> weaker acid
- Polar groups –> water loosely bound and proton mobility not impaired
5
Q
- Solubility - lipid media
A
- Generally peptides are polar –> poor lipid solubility
EXCEPT Cyclosporine (very hydrophobic): - cyclic peptide with N- C- terminal joined by amide bond –> can’t ionise in water
- methyl substitutions on amide N –> increase lipophilicity and decrease H Bonding with water
- Amino acids with hydrocarbon side groups
- Formulation requires micellar surfactant system
6
Q
- Isoelectric point
A
- Peptides have this –> pH value where absolute number of -ve charged groups equals absolute number of +ve charged groups
- mean value of all side chain pka but may change
At isoelectric point, molecule is electrically neutral with great tendency to self-associate (solid forming) by ionic interactions (of charged groups) and hydrophobic interactions (slow cos rearranging water takes time)
If pH further removed from isoelectric point (i.e. increased charge either +/-ve), there will be increased charge-dipolar interactions with water and increase coulombic repulsion between protein which increase aq solubility
If pH too far from isoel pt –> hydrolysis, denaturation and degradation of protein
7
Q
- Adsorption
A
- Proteins adsorb onto inert hydrophobic surfaces –> destabilise and denature protein
- Serum albumin, micellar surfactants used to compete for surface binding sites to reduce protein adsorption
8
Q
Preformulation - Stability aspects of peptides and proteins
A
Primary structure sequence - hotspots
- Hot-spot amino acids associated with amide bond instability e.g. Aspartic acid, asparagine, glutamic acid, glutamine
- hotspot contributors (a.a with short side chains) if placed immediately adjacent to hotspot amino acids on the C-terminal can enhance instability, due to lack of steric hindrance e.g. glycine, serine, alanine, proline, threonine
9
Q
Degradation Reaction Pathways
A
- nucleophilic substitution reaction reactions (hydrolytic instability)
Consequences: - change in overall electronic charge (isoelectric point)
- change in length and shape of peptide backbone through isomerisation
- proteolysis of peptide backbone into smaller fragments
Oxidation pathways occur in: Cysteine, methionine, tyrosine, tryptophan
THIS IS IMPORTANT IN DELIVERY MODE
10
Q
PREFORMULATION - Analytical detection of peptide or protein degradation products
A
To detect peptide/ protein degradation
- Electrophoresis methods
- charged molecules move at different rates in various aq media to separate mixture of molecules - High Performance Liquid Chromatography
- not preferred
- Using large pore diameter solid phases to separate molecules - Tryptic digestion followed by HPLC = peptide mapping
- Digest proteins with enzymes by cleaving at specific amino acid bonds to produce specific fragments which can be analysed by HPLC. –> generate peptide pattern to detect degradation even isomerisation - Immunological methods eg ELISA
- using antibodies to detect tiny quantities of protein - Spectroscopic and Thermal methods
- ORD/CD for 2 and 3 changes in structure
- NMR changes in structure, water content of protein and changes in functional group environment
- Mass Spec changes in molar mass
- Infrared detect changes in functional group and water content - Edman chemical degradation
- sequentially identify first 5-30 amino acids starting from N terminal - Ellman’s test
- detect free cysteine groups - X-ray crystallography
- protein structure and conformation in solid state
11
Q
PREFORMULATION - Stabilisation Methods
A
Lyophilizing the product (by freeze drying)
- to remove peptide/ protein contact with water
- avoid hydrolysis
Steps:
- Protein dissolved in suitable aq buffer solution
- Add excipients to protein solution
- Solution forced through sterile filter 0.22um
- Sterile solution distributed into sterile vials and partly capped with slotted rubber stoppers
- Solution frozen and exposed to vacuum
- Ice freezes then protein and excipients
- Sublimation to remove solid ice and using cold condenser to drive removal of water vapour
- Bulk ice removed, solid protein cake warmed to15-40 deg and residual water removed (2 deg drying)
- Slotted stoppers fully inserted into vials and over sealed with aluminium outer cover to seal the stopper
12
Q
Features of good freeze drying lyophilised protein
A
- no protein folding/ denaturation during freezing and water removal
- glass transition temp of final product is higher than intended storage temp
- low final water content
- elegant, strong, rapidly dissolving cake is formed
- chemical degradation has been limited
13
Q
Other physicochemical methods for stabilisation
A
- correct use of buffer and solution pH
- using antioxidants, chelators, complexing agents
- formulating to poorly water soluble, suspended fine particle solid
- adding surfactants
14
Q
Stabilisation by genetic manipulation of primary sequence
A
- If the amino acids causing instability are not part of the critical binding site, then these a.a can be replaced to improve stability by changing the DNA coding
Eg Human B-interferon
15
Q
Stabilisation by peptidomimetics
A
- Involves the design of a peptide that can perform a particular therapeutic task
- Then design a non-peptide analogue which has same polar, electronic and steric structure as the peptide inhibitor, so it can block the desired site of action
- These are designed without the potential for unstable peptide bonds that give easy degradation or digestion by GIT enzymes
