Protein Stability Oligonucleotide Stability

Question 1

What is the Drug Development Process defined according to what?

Answer 1

• What the regulatory agencies require e.g. FDA, TGA
• They all require:
  
  • Physical and chemical properties (e.g. if we don’t know solubility - can’t formulate with confidence)
  • PK properties (e.g. half life, distribution)
  • Analytical methods
  • Manufacturing methods
  • Animal, human studies
  • Documentation
  • Marketing approval

Question 2

Drug Development Process: What are the Important Physical and Chemical Properties?

Answer 2

• Melting point, boiling point
• pK_a
• Hygroscopicity
• Solubility
• Partition coefficient
• Stability (light, acid/base)
• Optical activity

Question 3

Protein Structure: What is the Primary Structure?

Answer 3

• Covalent backbone of polypeptide chain and sequence of amino acids

Question 4

Protein Structure: What is the Secondary Structure?

Answer 4

• Regular, recurring arrangement in space of the polypeptide chain along one dimension

Question 5

Protein Structure: What is the Tertiary Structure?

Answer 5

• Holy polypeptide chain is bent or folded in 3D

Question 6

Protein Structure: What is the Quaternary Structure?

Answer 6

• How the individual polypeptide chains having two or more chains are arranged in relation to each other

Question 7

Protein Structures: What does Confirmation refer to?

Answer 7

• More general term to refer to the combined secondary, tertiary and quaternary structure of protein

Question 8

Protein Structures: What does Oligomeric Proteins refer to?

Answer 8

• Proteins with one or more polypeptide chains - there component chains are called subunits or protomers
• e.g. haemoglobin

Question 9

What bonds do Protein Structures consist of?

Answer 9

• Electrostatic Interactions
• Hydrogen bonding
• Van der Waals forces
• Hydrophobic interactions
• Disulphide crosslinks

Question 10

Conformational Structure may be altered due to?

Answer 10

• pH changes
  
  • Changing pH can affect stability
  • e.g. pH too high or low in injectable - vein damage or discomfort
• Ionic strength changes
  
  • e.g. injectable product - adjust closer to isotonic to avoid stinging
• Surface active agents
  
  • SLS - added to optimal concentration and increase stability and solubility
• Shear stresses
  
  • Can occur if manufacturer has a tank that needs to pump to another tank that can cause enough shear to cause degradation
• Adsorption
  
  • e.g. molecule in solution, not soluble in water - stick to surfaces e.g. glass
• Disruption of water-protein interactions
  
  • These interactions can change depending on differences in concentration of the peptide within the solution

Question 11

What are Degradation Pathways?

Answer 11

• Hydrolysis, including deamidation
• Other deamidation reactions
• Transpeptidation
• Racemisation
• Oxidation
• Diketopiperazine formation
• Disulphide exchange
• Photodecomposition

Question 12

What is Native, Functional Protein?

What can happen to this protein in Solution?

Answer 12

• Any protein or peptide that might be in an injectable form or already circulating the body is a native, functional protein
  
  • It is active
  • Slowly degrading
• Rate of unfolding and refolding depends on pH, temperature
  
  • Reversible process, there’s equilibrium constant
  • In equilibrium with Partially or Fully Unfolded Protein

Question 13

What happens to the Partially or Fully Unfolded Protein?

Answer 13

• Noncovalent changes (Physical attraction in some way, not usually administered with other drugs)
  
  • Aggregation
    
    • molecule in solution, over time, slow unfolding and reversible process going on, a portion of the partially or fully unfolded protein may start to aggregate with itself - still remains in solution
    • Not active when in this form
  • Surface adsorption
    
    • The larger the molecule, the less soluble in water, the more likely the surface adsorption will be
  • Precipitation
    
    • Can happen because of aggregation - solution becomes cloudy
• Covalent Changes (irreversible)
  
  • Deamidation
  • Hydrolysis
  • Oxidation/photodecomposition
  • Racemisation
  • Disulphide exchange

Question 14

What are the important points of Asparaginyl Hydrolysis?

Answer 14

• All peptides are susceptible to hydrolysis
• Asparaginyl group most reactive
• Different mechanisms can occur
• The breakdown products are different and any of those breakdown products could be much less active in the patient

Question 15

How can we improve stability of Asparginyl Groups?

What is a problem with this?

Answer 15

• Could look at the amino acid sequence and replace glutamyl with glutamyl residue which can eliminate or reduce the side reaction which can increase the shelf-life
• However, if they change the sequence, it may change what happens at the receptor - may diminish activity

Question 16

Is Adrenocorticotrophic Hormone soluble in water?

Answer 16

• Freely soluble in water, partly precipitated at pI (4.65-4.8)
  
  • If we increase pH away from pI = overall negative charge on molecule
  • If we decrease pH away from pI = overall positive charge on molecule and therefore more soluble

Question 17

What are Disulfide Bonds?

Answer 17

• Most common encountered cross-link of proteins
• The only covalent link synthesised de novo in globular proteins
• Can be between two portions of a single chain or bridge between two otherwise independent chains
• Formation from oxidation of two cysteine residues

Question 18

Disulfide bonds: Cross-link formation - half-reaction for oxidation of cysteine to disulphide

Is this reaction reversible?

What does it form?

Answer 18

• It is reversible
• Form disulphide bridge

Question 19

Disulfide Exchange

What do the rates of forward and backward reactions depend on?

Answer 19

• Rates of forward and backward reactions are pH dependent since the thiolate ion participates
• Depending on the pKa of the thiols, electrostatic environment, position within folding, ring strain - can influence the rate of disulfide exchange occuring
• GSH has be used to help stabilise peptides and proteins

Question 20

What is a normal measure of Small Molecule Solubility?

Answer 20

• Excess solid in solvent at appropriate pH, stir it up for 24hrs (if stable), filter, take off the solid, take the filtrate, dilute it out to appropriate amount, then determine the concentration, then allow for the dilution factor, then they can work out solubility
• This is ok for small molecule but for a peptide or protein, as it gets more and more concentrated, just forms a gluggy mess

Question 21

How to Measure Protein Solubility?

Answer 21

• Different concentrations of PEG 8000 - dissolve in water
• Add the peptide and protein to the PEG
  
  • Easier to find a point where cloudiness starts = this is the maximum solubility
• Can use as little as 10mg of protein

Question 22

Protein Solubility: What can Organic Solvents do?

Answer 22

• Decrease dielectric constant
• Increase electrostatic attractions
• Decrease solute solubility

Question 23

Protein Solubility: What can Polyols and Sugars do?

Answer 23

• Can increase solubility
• Can increase stability at a higher concentration
• Trial and error at different pH, different concentrations, different excipients - determine what works best for solubility and stability

Question 24

List the processes of Protein Adsorption

Answer 24

• Adsorption to solid surfaces
• Solid/liquid interface
• Air/water
• Oil/water

Question 25

Chelating Agents (e.g. EDTA) - what is the role?

Answer 25

* Can decrease initiators of free-radical oxidation reactions * Heavy metals help to catalyse these healthy metal reactions * Reducing agents can reduce an oxidised drug * Oxidised compounds are more readily oxidised than the agents they are to protect

Question 26

What are Chain Terminators?

Answer 26

* Agents capable of reacting with radicals in solutions to produce a new species, a chain terminator radical, which doesn't re-enter the radical propagation cycle * The new radical may be intrinsically stable or may dimerise to form an inert molecule

Question 27

What are other strategies to help reduce oxidation?

Answer 27

* Adjust pH * Under nitrogen * Low rates of shear * Appropriate containers * Appropriate closures * Prevent exposure to light * Optimum storage temperature