Lecture 5 - Peptide and Protein Therapeutics 1

Question 1

What is proteins and peptide stability influenced by?

Answer 1

Choice of excipients
Storage conditions - liquid, frozen, lyophilized
Delivery route and vehicle

Question 2

Define primary protein structure

Answer 2

Order of amino acids in the proteins

Question 3

Define secondary protein structure

Answer 3

Formation of alpha helices and beta sheets

Question 4

Define tertiary protein structure

Answer 4

The overall 3D structure of a protein

Question 5

Define quaternary structure of a protein

Question 6

Name what protein folding is driven by

Answer 6

Hydrophobic interactions
Electrostatic - like charges repelling each other or like charges attracting each other to form pairs
H bonding - Inter and intramolecular
VDW forces
Steric effects - can’t have two bulky grouse occupying the same space
Hydration
Disulphide bridges - binds between thiol residues

Question 7

What are the issues with protein folding?

Answer 7

Conformational changes - formation of incorrect structures, aggregation
Chemical changes - example, hydrolysis, oxidation, deamination, glycation disulphide bond rearrangement
Break peptide backbone
Modification of important residues
Change Protein Shape - conformational changes and aggregations

Question 8

What is protein aggregation driven by?

Answer 8

Exposure of the hydrophobic residues in the proteins and these hydrophobic residues can interact with the hydrophobic residues on other unfolded proteins and then you get the formation of aggregates
Can grow and cause larger filaments or disordered aggregates which can become insoluble and form cloudy particles which float around in the protein formulation

Question 9

Name the operations that may denature or aggregate proteins

Answer 9

Freezing/thawing
Agitation (interfaces) - shaking solution will make it go cloudy as aggregates have formed - energy into the system - helps proteins unfold
Sonication - unfolding due to energy
Contact with silicone oil - exposed to more hydrophobic residues
Low or high pH - affects ionisation of the protein - repulsion of charged species leading to unfolding and aggregation
Low or high salt
Specific salts
Chemical changes
Heat - energy added

Question 10

What are the consequences of aggregation/denaturation

Answer 10

Altered solubility
Hypo-potency
Hper-potency
Off target binding - ADR, faster clearance
Patient may generate neutralising antibodies (ATAs) - makes drug ineffective, may break tolerance, cross-react with endogenous protein

Question 11

Name the chemical chnages to proteins

Answer 11

Deamidation
Oxidation
Hydrolysis (acid and base catalyst)
Disulphide formation and exchange

Question 12

Name the physical considerations for proteins

Answer 12

1) Temp - Tm at which 50% of the molecules are unfolded usually 40-80 degrees. Melting can lead to disaggregation, chemical reactions more rapid at increased temp. Thermophilic vs mesophilic proteins
2) pH
3) Adsorption and interfaces - air/water, organic solvents, vessels
4) Salts and metal ions - formulation and equipment
5) Concentration - increased conc - more likely to collide and aggregate

Question 13

Name the examples of different excipients added to the formulations of therapeutic proteins

Answer 13

Solubility enhancers - surfactants, amino acids, sugars, polymers
Antiadsorption and anti-aggregation agents - surfactants, albumin
Buffering agents - usually citrate, phosphate, acetate
Preservatives and anti-oxidants - ascorbic acid, antimicrobials
Lyoprotectants/cake formers
Osmotic agents - NaCl, mono- or disaccharides

Question 14

How do we avoid peripheral binding of solute?

Answer 14

Sugars, some amino acids - increased surface tension of water
Glycerols, polyols - repulsion - exclude solute molecules from the surface of the proteins
PEG - steric effects - exclude solute molecules from the surface of the protein

Question 15

Use of amino acids and proteins being added to formulation

Answer 15

Stabilise the formulation and prevent some chemical reactions from occurring
HSA - stabilises the protein
Methionine - oxidation conditions - will be preferentially oxidised
L-arginine and L-glutamic acid - interact with residues of opposite charges which may cause an association of proteins
Aliphatic regions can cover exposed hydrophobic areas of proteins

Question 16

Use of cylcodextrins (cyclic glucose molecules)

Answer 16

Outer hydrophilic surface and inner hydrophobic surface
Suppresses aggregation of proteins e.g. insulin and GH - hydrophobic pocket - prevents aggregation
HP-beta-CD approved for parental admin of leucine enkephalin

Question 17

Uses of polysorbates

Answer 17

Thought to stabilise proteins, preventing denaturation and aggregation via several mechanisms:
- preferential exclusion of solutes
- acting as a ‘chemical chaperone’ aiding protein refolding
- binding to hydrophobic patches of proteins
- formation of detergent film in aqueous systems to limit protein exposure to air/water interface
- compete with proteins in adsorbing to surfaces - glass, IV bags, tubing materials etc - reduces adsorption
- applicable for a lifetime of formulation from the manufacture to delivery

Question 18

Negative effects of polysorbates

Answer 18

Can undergo auto-oxidation and hydrolytic degradation - produce creative peroxides - W and M residues susceptible.
aldehydes - can react with K residues and N terminus, increased immunogenicity
formaldehyde and acetaldehyde cause contact allergies and are potential carcinogens

Antioxidants (including cysteine, glutathione, tryptophan, methionine) - can be added to reduce these effects
or degass any dissolved oxygen

Question 19

Extent of any affect with polysorbates depends on..

Answer 19

identity of the protein
protein and surfactant concentration
other excipients
temperature
light exposure

Question 20

Describe lyophilisation (freeze drying)

Answer 20

Low-temperature liquid phase - prolonged storage
freezing - better but their is issues - some salts crystallise more rapidly than others

Freeze drying:
- restricts mobility
- reduces RH
- allows RT storage

Question 21

Name the routes of administration

Answer 21

IV, SC, Intravitreal, buccal, pulmonary, others?

Question 22

Advantages of IV

Answer 22

Large doses can be administrated with 100% bioavailability
Administration can be controlled/ discontinued
Immediate access to the central compartment
Easy weight-based dosing

Question 23

Disadvantages of IV

Answer 23

Additional manipulation
Patient inconvenience/ compliance
The dose usually diluted into refilled IV bag - adsorption leading to lower concentration
Multiple materials of construction (polyolefin, PVC etc)
Agitation during transport may be significant
Risk of microbial exposure before use

Question 24

What can interfaces cause?

Answer 24

Aggregation

air/water - vials. IV bags - cause agitation
Oil-water - silicone coated syringes - cause aggregation and adsorption
Hydrophobic surfaces - IV set and bag - PVC ver polyolefin - cause aggregation and adsorption
care must be taken to minimise interfaces X

Question 25

Advantages of subcutaneous

Answer 25

Patient convenience/compliance
May require no compounding
Can incorporate an autoinjector
Best if flat dosing but can accommodate weight based

Question 26

Disadvantages of subcutaneous

Answer 26

The maximum dose is lower - max volume is lower than IV, new larger volume SubQ delivery options,20mL
Cannot stop dosing once administrated
Bioavailability is <100%

Question 27

Advantages of intravitreal

Answer 27

Direct site of action - 100% bioavailability

Question 28

Disadvantages of intravitreal

Answer 28

Patient compliance/convenience
Some risk of infection - immunologically privileged

Question 29

Advantage of buccal

Answer 29

Patient compliance/convenience

Question 30

Disadvantages of buccal

Answer 30

Drug loss < 100% bioavailability
Variability

Question 31

Advantages of pulmonary delivery

Answer 31

Local delivery, local high conc

Question 32

Disadvantages of pulmonary delivery

Answer 32

Nebulisers are typically large and bulky
Proteins not stable in organic solvents
Not all nebulisers are the same
Particle size distribution
Shear or air0water interface denaturation
Testing required for each nebuliser