Physical Stability

Question 1

What is the Primary Structure of Proteins?

Answer 1

• The sequence of amino acids that form the protein

Question 2

What is the Secondary Structure of Proteins?

Answer 2

• Regular repeated patterns of initial folding that are found in the protein
  
  • Alpha helices
  • Beta sheets

Question 3

What is the Tertiary Structure of Proteins?

Answer 3

• The shape of the protein when all the amino acids, alpha helices and beta sheets are folded into a specific structure

Question 4

What is the Quaternary Structure of Proteins?

Answer 4

• Protein that’s composed of two folded amino acid chain subunits that come together to form one final protein

Question 5

Discuss the Characteristics of the a-helix

• Why does the a-helix form?
• What is the interaction?
• What way do the -NH groups point?
• What way do the -C=O groups point?
• Why do these groups point the way that they do?

Answer 5

• The a-helix forms because -NH and -C=O groups interact through hydrogen bonding pulling the backbone into a spiral
• All the -NH groups point up
• All the -C=O groups point down
• To allow the formation of -C=O…H-N- hydrogen bonds

Question 6

What are the Characteristics of the B-Sheet?

• What does the B-sheet consist of and how are they connected?
• What is a B-sheet made up of?
• What formation can the B-sheet be?
• How are they normally represented?

Answer 6

• The beta sheets consist of b-strands, connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet
• A b-strand is a polypeptide chain typically 3-10 amino acids long with backbone in an extended conformation
• Can have both ‘parallel’ and ‘anti-parallel’ beta sheets
• They are normally represented in protein topology diagrams by an arrow pointing toward the C-terminus

Question 7

Characterising Protein 2° Structures: What is used in determining protein structure?

What absorb in this energy range?

Answer 7

• Far-UV CD
• Amide bond electrons absorb in this energy range
• Overall, there’s different spectra depending on whether we have beta sheet or helices

Question 8

Are Proteins Soluble?

Are amino acids can be what?

What happens as pH moves away from the isoelectric point?

Answer 8

• Proteins can range from very soluble to virtually insoluble
• Amino acids may be acidic/basic or hydrophilic/hydrophobic
• Solubility of globular proteins increases as pH moves away from the isoelectric point

Question 9

What is Infliximab? How does it act

What is TNF-a an important part of?

Answer 9

• Infliximab is a mab against TNF-a, it acts by binding to TNF-a
• TNF-a is an important part of the autoimmune system - a chemical messenger (cytokine)

Question 10

What is an Autoimmune disease?

Answer 10

1. When the immune system mistakenly attacks and destroys healthy body tissues

Question 11

Formulating Biosimilars

• Are polypeptides stable?
• Are polypeptides potent?
• Do preparations need to be sterile?
• How can the active be lost?
• How can bioactivity be preserved?

Answer 11

• Polypeptides are inherently unstable
• Polypeptides are highly potent, hence are used at much lower concentrations than traditional ‘small molecule’ therapeutics
• Preparations must be sterile
  
  • This may require the use of filters - use low protein binding filters to minimise loss of active
• Will also lose active by adsorption of proteins to containers
• Incorporate various stabilisers in formulations, to preserve bioactivity

Question 12

Formulating Biosimilars: Additional Processing Steps often used for Biologics

What is Lyophilisation (Freeze Dried)?

Answer 12

• Gently removing water from system
• Additional excipients required to prevent the formation of large insoluble protein aggregates

Question 13

Formulating Biosimilars: Additional Processing Steps often used for Biologics

What is PEGylation?

Answer 13

• Chemical modification of the polypeptide, by chemically bonding to PEG, improving the stability of the protein
• Make more stable - can make more resistant to aggregation when in aqueous solution
• Don’t add PEG to any active site on protein, may lose some fraction of proteins

Question 14

Manufacturing Biosimilars

• Are biopharmaceuticals easy to manufacture?
• What does the use of living organisms introduce?
• What is required to guarentee the quality and compliance of each production batch?
• What can have major clinical consequences?

Answer 14

• Biopharmaceuticals are difficult to manufacture due to their structural complexities
• The use of living organisms introduces an inherent variability in the manufacturing process
• To guarantee the quality and compliance of each production batch, absolute consistency of the manufacturing process is required
• Even apparently slight changes in any of the manufacturing or formulation steps can have major clinical consequences

Question 15

Protein Stability: What is there a concern with?

Answer 15

• Protein aggregation and instability can be cause of many disease states
  
  • Impact on multiple feedback mechanisms so we get damaged proteins in body
• Protein misfolding diseases are found in multiple organs and can be defined histopathologically by the presence of specific misfolded protein(s) deposits
  
  • Misfolded protein diseases often feature complex interactions between aggregates of multiple proteins - all misfolded proteins share a common structural feature, known as the ‘amyloid fold’

Question 16

What is Protein Denaturation?

Denaturation is reversible if?

Denaturation is irreversible if?

Answer 16

• Loss of polypeptide’s specific 3D conformation, typically by disruption of secondary/tertiary/quaternary structures
• Denaturation is reversible if the native conformation can be regained
• Denaturation is irreversible if the native conformation is permanently lost

Question 17

What can Denaturation occur due to?

Answer 17

• Increase in temperature
• Protein adsorption at interfaces
• Presence of small molecules

Question 18

Denaturation is controlled by formulation including:

Answer 18

• Inclusion of surface-active species
  
  • Prevent protein aggregation
  • Prevent protein adsorption at interfaces
• Addition of cosolvents
• Buffering
• Chemical modification

Question 19

Protein Aggregation: Characterisation

• What do particles in suspension undergo?
• What are advantages of Dynamic Light Scattering (DLS)?

Answer 19

• Particles in suspension undergo Brownian Motion, due to solvent molecular collisions in random thermal motion related to:
  
  • Size, viscosity, temperature
• Advantages:
  
  • Non-invasive measurement
  • Can measure low sample quantities
  • Can measure concentrated samples
  • Capable of detecting trace amounts of aggregation

Question 20

Dynamic Light Scattering (DLS)

• What is it?
• In brief what happens to large and small particles?
• What are the limitations?

Answer 20

• Autocorrelator converts intensity fluctuations signal into particle size distribution
• In brief
  
  • Large particles move slowly in solution
  • Small particles move more quickly
• Limitations
  
  • Intensity based measurement - large particles scatter more light than smaller particles which complicates measuring mixed samples