protein preparations Flashcards
What are proteins composed of?
amino acids (base) linked by amide bonds
fibrous and globular
examples of fibrous proteins
mechanical functions
- hair
- skin
- bones
- connective tissue
examples of globular proteins
enzymes
antibodies
inhibitors
How many AA does insulin have?
55 AA (peptide)
What formulations are proteins given in?
injection
How are therapeutic proteins formed?
extracted from natural sources
OR
engineered in a lab
- recombinant deoxyribonucleic acid (rDNA) technology or gene cloning
examples of biopharmaceutical proteins
vaccines recombinant proteins blood products enzymes mAb nucleic acid-based therapeutics
Brand name for recombinant human insulin
Humulin
What challenges are there with proteins/insulin?
producing stable and biological active therapeutic protein for LT storage
When AA join what do they form?
proteins
How do AA join?
by formation of a peptide bond
1 molecule of water is released (condensation)
COOH—NH2
What are peptide bonds susceptible to?
hydrolysis
What is primary structure of a protein?
linear sequence of AA
What is secondary structure of proteins?
polypeptide chain folds and turns by H bonding
-> gives alpha helices and beta sheets
What is tertiary structure of proteins?
folded, native or 3D structure
- biologically active in this shape
- protein can have biological effect
- polypeptide chains become functional
What is quaternary protein structure?
Ig or antibody structures (large molecules)
several protein chains packed together
held together by H bonds, van der Waals forces
chemical instability of folded/native proteins
deamidation (Glu, Asp)
oxidation (His, Met, Cys)
peptide bond hydrolysis
disulfide exchange
-> leads to irreversible denaturation
physical instability of folded/native proteins
temperature pressure pH (ionisation) surface adsorption (contsiner/package) aggregation
-> reversible denaturation
challenge with proteins - aggregation
aggregation is a problem in high conc formulations
may lead to loss of efficacy/safety
based on surrounding environmnt (pH, ionic strength, temp, excipients, stress)
deatured proteins
unfolded
not active
What happens if you remove water from proteins?
forms dry proteins
inaccurate native structure due to protein protein interactions
-> not active if dry, need water (reconstitute back to native structure)
Why are surfactants used in protein formulations?
act as a stabiliser during processing to prevent destruvtive effect of heat/shear
Whay are sugars added as an excipient?
sugars prevent interactions between protein molecules
help proteins stay in native structure
2 excipients used to protect proteins from air-liquid interface effect
sugars
surfactants
preferential hydration of proteins
water molecules have higher affinity for protein than excipients
What is preferential binding of proteins?
excipients have higher binding to the surface of the protein
excludes water
How to make proteins stable?
co-solute omitted from surface of protein
interactions with water molecules more desirable than interactions with additive
formation of a ‘hydration shell’ around protein required for protein activity
What is a ‘hydration shell’?
water molecules interact with protein surface and bind
more desirable than interaction with additive
shell required for protein activity
How are proteins in liquid form given?
injections
solutions for nebulisation
What maintains protein stability in liquid form?
additives
examples of additives
sugars polymers cyclodextrins salts (pH, ionic strength) non-ionic surfactants
examples of sugars used as additives
trehalose
mannitol
sucrose
What pH is insulin soluble at?
pH < 5
What form of proteins is more stable for LT storge?
solid forms
excipients in powder protein formulations
bulking agents (trehalose, lactose)
salt (NaCl)
properties of injection formulations
sterile
isotonic
clear after reconstitution
low viscosity
What techniques are used to produce proetins in dried powder forms?
freeze drying
spray freeze drying
spray drying
supercritical fluid technology
difference between freeze drying and spray freeze/spray/supercritical fluid tech
freeze drying - cannot produce dried protein particles with controlled particle size
the others - can produce protein particles with controlled particle size (< 5 micro m)
What can spray drying modify?
particle size and shape
particle surface texture
What does spray drying do?
- particles dissolved/suspended in a liquid
- converts contents of solution/suspension into powder in 1 step
steps of the spray drying process
- atomisation of the liquid as fine droplets into a hot air stream by spray nozzle
- atomisation creates a large SA and rapid evaporation of the solvent
- after droplets dry, the powder is separated from the air stream by cyclone separator
What properties of the drying chamber affect droplet stability?
protein denaturation
water content
hot gas in contact with protein droplets
What properties of the air intake affect droplet stability?
pH, protein, additives concs
flow rate
temperature
What properties of the dry product affect droplet stability?
LT stability
packaging
storage conditions
particle shape
What spray drying variables affect the resulting particle size?
- spray pattern
- conc of solute
- air flow system
Why is protein denaturation minimal?
cold water is circulating around the protein solution
AND
presence of protein solution droplets in drying chamber is for a short time
What types of particles can spray drying produce?
solid particle
hollow particle
cenosphere
What additives are good/bad to stabilise proteins?
good - poly vinyl pyrrolidone
bad - methacrylic acid (destabilises freeze dried proteins)
What additives are good/bad to stabilise proteins?
good - poly vinyl pyrrolidone
bad - methacrylic acid (destabilises freeze dried proteins)
What is Circular Dichroism used for?
characterises secondary and tertiary structure of proteins in solutions
