ILRA stabilisation - biologics

Question 1

a) Explain why the rate of ILRA degradation increased with increasing temperature and what effect this would have on the ILRA protein structure.

Answer 1

• Increase of temperature causes the protein to move more and more likely to collide and form aggregates. Also, higher temp makes It more likely to unfold and denature.
• An increase of the physical and/or chemical degradation of the protein under heat stress is observed and may lead to instability/exposure of hydrophobic region.
• Together with increased collision of solvent / solute molecules at higher temperatures, increased conformational change and (partial) unfolding of the protein, results in aggregation & increased turbidity (note: turbidity measures the presence of particles as they scatter more light but is also observed at high concentration: thus only the change of turbidity is relevant).

Question 2

Compare the rates of degradation and rates of increase in turbidity at the different temperatures at low sucrose concentrations, and explain why degradation may have led to increased turbidity.

Answer 2

Importance of sucrose:
• Degradation rates higher at low sucrose concentration than the rates of increase in turbidity
• Greater protein conformational change and unfolding would lead to exposure of more hydrophobic regions buried in the interior of the protein.
• Increased hydrophobic exposure leads to bonding/attraction between (partially) unfolded proteins may lead to aggregation , increase in particle size and turbidity.

Question 3

c) Compare and explain why the degradation rates were lower at higher sucrose concentrations

Answer 3

• Higher sucrose concentrations = stabilises protein
• Sucrose hydrophilic – attracts water molecules from protein, protein shrinks = mpre stable and less likely to unfold.
• Lower rates of degradation and less turbidity observed at higher sucrose concentrations, with small/insignificant rates at 8°C and 30°C, but with a significant basal rate at 50°C unchanged by sucrose concentration.

The more compact and tightly folded protein conformation at higher sucrose concentrations may be more resistant to unfolding and side chain reactions at higher temperature.
Consequently, formation of large aggregates is less favoured at higher sucrose concentrations. This may be related to the increased chemical potential and surface area of more unfolded conformations and denatured states.

Question 4

d) Explain how the concept of preferential exclusion may apply

Answer 4

• Sucrose (hydrophilic – attracts water molecules) stabilising excipient is preferentially excluded from the surface of the protein, which increases the chemical potential or energy of the unfolded state.
• The degree of preferential exclusion and the increase in chemical potential are directly proportional to the surface area of protein exposed to solvent.
• System will minimise thermodynamically unfavourable effects of preferential exclusion of sucrose by favouring the state with the smallest surface area / lower chemical potential / native state.
• As the unfolded / denatured state will have a higher surface area, and increased chemical potential, with preferentially excluded sucrose, the free energy of denaturation is increased, when the native state would be favoured thermodynamically.
• Based on this thermodynamic principle, increased sucrose should also favour the most compact protein conformation, even under non-denaturing conditions, when the protein would expected to fold more tightly.
• A more compact/tightly folded conformation would be expected to be of smaller apparent size, when increases in sucrose concentration would be expected to result in more compact conformations and smaller apparent protein size.

Question 5

Preferential interaction & exclusion:

Denaturant

Answer 5

interaction with polypeptide backbone of protein e.g. urea with most amino acid side chains (e.g. by H bonding)
• higher interaction when unfolded +ve concentration difference between local and bulk domain.

Question 6

Preferential interaction & exclusion:

Protectant

Answer 6

e.g. sucrose osmolyte strong interaction with water
• lower interaction (e.g. hydrophobic) & higher exclusion when unfolded  -ve conc difference between local & bulk.

Question 7

What is the double funnel model?

Answer 7

• The double funnel model describes the conformational space of selection of folding or aggregation from intermediate states, whose architecture depends on environmental conditions.
• Aggregation funnel is less jagged than the native funnel, due to lower restrictions on aggregate conformations.

Question 8

Low temperatures (stabilisation)

Answer 8

• Low temperatures extend shelf life, cold denaturation is often reversible.
– As temperature drops, properties of solvent change including dielectric constant, acid/base ionization, diffusion rates, solubility of hydrophobic residues and hydrogen bond energies
• Freezing provides lower temperatures BUT repeated cycles of freezing and thawing cause aggregation by pH and concentration changes and provision of nucleation points on the surface of ice-water interfaces.

Question 9

How does cryoprotection work?

Answer 9

• Cryoprotection by sugars, polyhydric alcohol, oligosaccharides, amino acids, surfactants.
• Largely work by preferential exclusion, lowering cold denaturation temperature and stabilising osmotic stresses, whereas surfactants interfere with ice / water interface.

Question 10

A frozen vial of a liquid protein biologic product needs to be dispensed into an IV infusion bag. Which ONE of the following do you think would dispense the product as safely as possible?

A. Thaw sufficient liquid product from the frozen vial to dispense into infusion bag and return the remaining product to freezer.
B. Weigh out the required frozen product, dispense into the infusion bag and return remaining frozen product to the freezer.
C. Thaw and shake well in the vial before dispensing into the infusion bag and discarding remaining product.
D. Thaw and swirl the liquid product before dispensing into the infusion bag for fridge storage for up to 24h until needed, and discard remaining product.
E. Thaw and squirt the liquid product from a syringe back into the vial a few times to mix before dispensing into the infusion bag, and return remaining product to the freezer.

Answer 10

D. Thaw and swirl the liquid product before dispensing into the infusion bag for fridge storage for up to 24h until needed, and discard remaining product.

Question 11

Why do vials need to be mixed?

Answer 11

Concentration gradients are formed during freezing, and tend to remain, if thawing performed without mixing. Vial shape, size & materials affect freezing pattern.

(Shaking imposes a lot of stress to protein – could cause protein to unfold therefore mix GENTLY)

Question 12

Case study: polymer implant somatotrophin – explain losses during production & use

Answer 12

• More stable in dry state than dilute solution on release, but higher losses during dehydration and rehydration of implant (similarly in freezing and freeze drying)
• Dehydration concentrates excipients and protein, also resulting in pH change (depending whether predominance of acidic or basic ionizing groups)
• Change in pH & increase in ionic strength (electrostatics), surfaces etc make unfolding more likely  increase in surface hydrophobic regions (viz reverse phase RP- HPLC detection)
• Greater concentrations increase the probability of collisional interactions  increase in aggregates (viz size exclusion SE-HPLC detection)
• Protein goes through this twice upon manufacture / drying and similarly again on use / rehydration .