Protein Preparations Flashcards
what are proteins?
Proteins are composed of amino acids (BASE)
linked by amide bonds. There are 20 known amino
acids: L amino acid is of biological origin.
how are proteins classified?
hey are
classified as Fibrous proteins and globular proteins.
Examples for globular proteins include enzymes,
antibodies, inhibitors. Fibrous proteins provide
mechanical functions: hair, skin, bones and
connective tissues.
how many amino acids does insulin have?
51
how can therapeutic proteins be made?
can be either
extracted from natural sources or can be laboratory
engineered for pharmaceutical uses
Recombinant
deoxyribonucleic acid (rDNA) technology or gene cloning.
what are examples of biopharmaceutical proteins?
include vaccines,
recombinant proteins, blood products (e.g. Recombinate®
factor VIII), enzymes, monoclonal antibodies and nucleic
acid-based therapeutics.
how is the protein conformational structure made up?
primary structure- It is an identification for protein chemical properties, it is the linear sequence of amino acids. 2- Secondary structure Polypeptide chain folds and turns by hydrogen bonding: α-helices and β- sheets 3- ffolded, native or 3D 4- quaternary sturcture
what are the challenges facing formulations of therapeutic proteins?
chemical instability -deamidation (Glu and ASP), oxidation (His, Met, Cys), peptide bond hydrolysis, disulfide exchange this leads to irreversible denatureation
physical instability-temperature; pressure; pH (ionization), surface adsorption, aggregation this is reversible
what are the structure complexity challenges for manufacturing proteins?
Aggregation is a problem particularly in high
concentration formulations:
May lead to loss of efficacy and safety
Based on surrounding environment: pH, ionic
strength, temperature, excipients and stress.
Understanding the mechanism may help
formulations and selection of processing.
when is a protein stable?
when it is in its native state
what happens if you remove water
dry proteins….inaccurate native
structure due to protein protein interactions
what does the addition of sugars do to the protein?
Prevent by some way this interactions (in
solid state stabilisation)
what use are surfactants during protein stabilisation?
Surfactants used as stabiliser during processing to
prevent destructive effect of heat or shear. Also, they
can protect proteins from air-liquid interface effect
when is a protein stable?
Thermodynamically stable, folded
conformation
why is the co-solute omitted from the surface of proteins ?
because the interactions of the surface with water
molecules are more desirable than between
interactions of the protein surface with the additive
and this will lead to formation of a “hydration shell”,
around the protein, which is required for protein
activity.
what are the methods for protein preparation?
in liquid form- contains additives to maintain protein stability
what are examples of different additives?
Proteins are unique i.e. additives work differently with different proteins
do additives work the same?
additives work differently with different proteins
how was dry powder insulin made up?
- Bulking agent e.g. trehalose, lactose
- Salt e.g. NaCl
- Modification of the surface.
what is required for injections in the form of powders to be reconstituted?
Sterile
- Isotonic ….salts
- Clear after reconstitution…trehalose, glycine
- Low viscosity …..surfactant
what are the techniques for prep of porteins in dried powders?
➢Freeze drying –can not produce dried protein particles
with controlled particle size, hence it is a common
technique for protein powder formulations to be
reconstituted before injections
➢ Spray freeze drying
➢Spray drying
➢ Supercritical fluid technology
The last three mentioned techniques can produce protein
particles with controlled particle size (less than 5mm)
how can spray drying modify the powder properties?
▪Particle size and shape
▪Particle surface texture
how does spray drying work?
n spray drying, the original particles are dissolved
or suspended in a liquid.
❖Spray drying converts the contents of the solution
or suspension into powder in one step.
what is the process of spray drying?
▪ Atomisation of the liquid as fine droplets into a hot air stream (in a chamber) by a spray nozzle
Figure 1: A diagram of a spray drying system: 1-liquid feed, 2-air intake,3-atomiser, 4-drying chamber, 5-dry product,6-air outlet, 7-cyclone
▪ Atomisation creates a large surface area and very rapid evaporation of the solvent
▪ After drying of the droplets, the dried powder is separated from the air stream by a cyclone separator
what stress occurs in spray drying?
shear stress
what are the variables in spray drying that affect the particle size?
▪ The spray pattern
▪The concentration of the solute (s)
▪The air flow system
how can protein denaturation occur in spray drying?
Protein denaturation due to the heat effect in
the spray drier is to the minimal, because cold
water is circulating around the protein solution
and the presence of protein solution droplets in
drying chamber is for only short time.
what are the 3 forms that the particles can be spray dried?
solid
cenosphere
hollow particle
how are spray dried proteins characterised?
Microscopy Particle size Water content Thermal stability Secondary structure Density
how can proteins be stabilised?
It was found that:
- Poly vinyl pyrrolidone can stabilise both proteins,
however,
- Methacrylic acid destabilises both spray dried
proteins –it was not a good choice of additives
what are examples of characterisation tests?
- Circular Dichroism: for both secondary structure
and tertiary structure of proteins in solutions - Biological activity: for tertiary, folded and active
structure of the proteins - Differential scanning calorimetry: for thermal
stability of protein formulations
