Lec 3- Biotechnology-based pharmaceuticals Flashcards

1
Q

Proteins v peptides

A
  • 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
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2
Q

Peptides, Proteins and delivery routes

A
  • 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
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3
Q

Stability of biotech pharmaceuticals

A
  • 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
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4
Q

Protein structure

A
  • Protein structure is not a simple matter
  • Unlike traditional, low molecular weight drugs, protein therapeuticsz neeed to be stabilised in 4 levels
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5
Q

Protein structure levels

A
  • 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
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6
Q

Physicochemical properties of proteins in solution

Hydrodynamic properties

A
  • A folded protein is more compact than an unfolded one
    • This results in lower solution viscosity and generally faster rate of sedimentation
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7
Q

Chemical reactivity

A
  • There is a great variety in chemical reactivity of functional groups
    • Unreactive: Hydrocarbon side chain
    • Reactive: Cysteine groups
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8
Q

Water solubility

A
  • 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)
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9
Q

Protein instability

A
  • Degradation for proteins can be categorized into 2 distinct classes
    • A) Chemical instability (irreversible)- covalent changes
    • B) Physical instability (often reversible) - non-covalent changes
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10
Q

Mechanisms involved in the degradation of protein pharmaceuticals

A
  • Native functional proteins =>
    • Paritally or fully unfolded proteins =>
      • Non-covalent changes- Aggregation; surface adsorption; precipitation
      • Covalent changes- Hydrolysis; deamindation; oxidation; racemization; disulphide exchange
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11
Q

Denaturation

A
  • 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
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12
Q

Denaturation

Factors and outcomes

A
  • 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
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13
Q

Stability problems observed with proteins

A
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14
Q

Formulation strategies

A
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15
Q

Protein stabilisation in the dried solid state

A
  • 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
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16
Q

Formulation strategy: Excipients

A
  • Excipients can be essential during lyophilisation to protect the integrity of the protein, during reconstitution and stabilising the protein in solution
17
Q

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

1) MW

A
  • 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
18
Q

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

2) Ionic charge

A
  • Cationic polymer bind anionic proteins or peptides to anionic tissues
  • Lysine, Arginine and histidine all have basic NRH+ side chain
19
Q

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

3) Molecular specificity

A
  • 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