Protein Preparations

Question 1

what are proteins?

Answer 1

Proteins are composed of amino acids (BASE)
linked by amide bonds. There are 20 known amino
acids: L amino acid is of biological origin.

Question 2

how are proteins classified?

Answer 2

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.

Question 3

how many amino acids does insulin have?

Answer 3

51

Question 4

how can therapeutic proteins be made?

Answer 4

can be either
extracted from natural sources or can be laboratory
engineered for pharmaceutical uses
Recombinant
deoxyribonucleic acid (rDNA) technology or gene cloning.

Question 5

what are examples of biopharmaceutical proteins?

Answer 5

include vaccines,
recombinant proteins, blood products (e.g. Recombinate®
factor VIII), enzymes, monoclonal antibodies and nucleic
acid-based therapeutics.

Question 6

how is the protein conformational structure made up?

Answer 6

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

Question 7

what are the challenges facing formulations of therapeutic proteins?

Answer 7

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

Question 8

what are the structure complexity challenges for manufacturing proteins?

Answer 8

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.

Question 9

when is a protein stable?

Answer 9

when it is in its native state

Question 10

what happens if you remove water

Answer 10

dry proteins….inaccurate native

structure due to protein protein interactions

Question 11

what does the addition of sugars do to the protein?

Answer 11

Prevent by some way this interactions (in

solid state stabilisation)

Question 12

what use are surfactants during protein stabilisation?

Answer 12

Surfactants used as stabiliser during processing to
prevent destructive effect of heat or shear. Also, they
can protect proteins from air-liquid interface effect

Question 13

when is a protein stable?

Answer 13

Thermodynamically stable, folded

conformation

Question 14

why is the co-solute omitted from the surface of proteins ?

Answer 14

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.

Question 15

what are the methods for protein preparation?

Answer 15

in liquid form- contains additives to maintain protein stability

Question 16

what are examples of different additives?

Answer 16

Proteins are unique i.e. additives work differently with different proteins

Question 17

do additives work the same?

Answer 17

additives work differently with different proteins

Question 18

how was dry powder insulin made up?

Answer 18

• Bulking agent e.g. trehalose, lactose
• Salt e.g. NaCl
• Modification of the surface.

Question 19

what is required for injections in the form of powders to be reconstituted?

Answer 19

Sterile

• Isotonic ….salts
• Clear after reconstitution…trehalose, glycine
• Low viscosity …..surfactant

Question 20

what are the techniques for prep of porteins in dried powders?

Answer 20

➢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)

Question 21

how can spray drying modify the powder properties?

Answer 21

▪Particle size and shape

▪Particle surface texture

Question 22

how does spray drying work?

Answer 22

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.

Question 23

what is the process of spray drying?

Answer 23

▪ 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

Question 24

what stress occurs in spray drying?

Answer 24

shear stress

Question 25

what are the variables in spray drying that affect the particle size?

Answer 25

▪ The spray pattern
▪The concentration of the solute (s)
▪The air flow system

Question 26

how can protein denaturation occur in spray drying?

Answer 26

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.

Question 27

what are the 3 forms that the particles can be spray dried?

Answer 27

solid
cenosphere
hollow particle

Question 28

how are spray dried proteins characterised?

Answer 28

Microscopy
Particle size
Water content
Thermal stability
Secondary structure 
Density

Question 29

how can proteins be stabilised?

Answer 29

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

Question 30

what are examples of characterisation tests?

Answer 30

• 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