Lec 3- Biotechnology-based pharmaceuticals

Question 1

Proteins v peptides

Answer 1

• A matter of size generally
• Peptides are compounds consisting of 2 or more amino acids
• Manufactures by either solution or solid phase peptide sysnthesis
• Protein are chains of 50 or more amino acids
• Manufactured by biotech methods

Question 2

Peptides, Proteins and delivery routes

Answer 2

• Currently there are only 2 peptides in oral formulations currently approved by the FDA
• Desmopressin
• Cyclosporine
• Look up both of these products, see their clinical use and consider their oral bioavailability

Question 3

Stability of biotech pharmaceuticals

Answer 3

• A pharmaceutical protein product should be stabilised against
  
  • Unfolding
  • Aggregation and self-association
  • Denaturation
  • Chemical degradation e.g. oxidation
• As well as
  
  • Changes in the amino acid sequence due to mutation occurring during fermentation

Question 4

Protein structure

Answer 4

• Protein structure is not a simple matter
• Unlike traditional, low molecular weight drugs, protein therapeuticsz neeed to be stabilised in 4 levels

Question 5

Protein structure levels

Answer 5

• Primary: Refers to the linear amino acid sequence and position of the disulphide links
• Secondary: Defines the spatial relationship of the amino acid residues e.g. a-helix or b-pleated sheets
• Tertiary: This takes into account the extent and nature of stabilising side-chain interactions (H-bonding)
• Quaternary: refers to the spatial relationship between polypeptide subunits of a protein

Question 6

Physicochemical properties of proteins in solution

Hydrodynamic properties

Answer 6

• A folded protein is more compact than an unfolded one
  
  • This results in lower solution viscosity and generally faster rate of sedimentation

Question 7

Chemical reactivity

Answer 7

• There is a great variety in chemical reactivity of functional groups
  
  • Unreactive: Hydrocarbon side chain
  • Reactive: Cysteine groups

Question 8

Water solubility

Answer 8

• Charged groups in proteins can increase solubility
• However ion-ion bond can form within the protein structure
• This neutralises the charge and influence solubility (As can electrolytes)
• Unfolding of a protein structure will also influence solubility
• When sufficient water solubility is lost, protein precipitation results (with loss of biological activity)

Question 9

Protein instability

Answer 9

• Degradation for proteins can be categorized into 2 distinct classes
  
  • A) Chemical instability (irreversible)- covalent changes
  • B) Physical instability (often reversible) - non-covalent changes

Question 10

Mechanisms involved in the degradation of protein pharmaceuticals

Answer 10

• Native functional proteins =>
  
  • Paritally or fully unfolded proteins =>
    
    • Non-covalent changes- Aggregation; surface adsorption; precipitation
    • Covalent changes- Hydrolysis; deamindation; oxidation; racemization; disulphide exchange

Question 11

Denaturation

Answer 11

• Refers to alteration of the global fold of a native molecule
• I.e disruption of the teritary and frequently the secondary structure
• Essentially changes the physical structure of the protein

Question 12

Denaturation

Factors and outcomes

Answer 12

• Factors
  
  • Temperature
  • pH
  • Addition of organic solutes e.g. urea, acetamide and formamide
  • Addition of organic solvents e.g. alcohols, acetone
• Outcomes
  
  • Protein becomes insoluble in aq solution (precipitation)
  • Loss of biological activity
  • Changes in molecular shape
  • Susceptibility to enzymatic hydrolysis

Question 13

Stability problems observed with proteins

Answer 13

Question 14

Formulation strategies

Answer 14

Question 15

Protein stabilisation in the dried solid state

Answer 15

• If the solutions are not chemically and physically stable for 1 year or more in solution then lyophilization (freeze drying= using pressure to make product solid without using pressure) can be used
• These techniques dehydrate the protein and inhibit the degradation reactions observed with proteins in solution

Question 16

Formulation strategy: Excipients

Answer 16

• Excipients can be essential during lyophilisation to protect the integrity of the protein, during reconstitution and stabilising the protein in solution

Question 17

Parameters engineering to control site-specific delivery of proteins and peptides

1) MW

Answer 17

• Low MW or cyclic peptides are more readily transported across biological membranes
• Conjugation of PEG to peptides and proteins enhances circulation and half-life
• PEG-L-asparaginase, PEG-Adenosine-Deamidase are now commercially available
• PEG-Interleukin-2 and other are clinical trials

Question 18

Parameters engineering to control site-specific delivery of proteins and peptides

2) Ionic charge

Answer 18

• Cationic polymer bind anionic proteins or peptides to anionic tissues
• Lysine, Arginine and histidine all have basic NRH+ side chain

Question 19

Parameters engineering to control site-specific delivery of proteins and peptides

3) Molecular specificity

Answer 19

• Chimeric fusion proteins combine the pharmacological activity of proteins and peptides with targeting moieties
• E.g. Transferrin receptors proteins and peptides across the blood-brain barrier