Unchained Labs eBook - 19

Unleash your biologic * See stability in hi-def with Hunky
A

B

Ideally stability is optimized by maximizing ΔG since the unfolded
state is often highly prone to aggregation. Aggregates can serve
as a thermodynamic sink, shifting the equilibrium away from the
stable native state, creating a risk for long-term stability. Reducing the amount of denatured protein in the sample by maximizing
ΔG can reduce aggregation risks, but this provides only a partial
look at aggregation. To determine aggregation propensity and
aggregation pathway, ΔG must be measured at low and high
protein concentrations.

Figure 4. If aggregates are present, ΔG changes as a function of protein
concentration. The aggregation pathway is determined by an increase
or decrease in ΔG at higher concentration with the 2-point Agg Path
experiment (A) or the detailed ΔGtrend experiment (B).

Figure 5. Aggregation can act
as a thermodynamic sink,
shifting the equilibrium away
from the desired native state.

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GENengnews.com

Protein unfolding is a unimolecular, concentration-independent
process in the absence of aggregation. In this case ΔG will not
change as protein concentration is increased. If aggregation is
present the measured ΔG values will display protein concentration dependence as equilibrium is shifted away from the desired
native state.2 The direction of change, positive or negative, is
indicative of the aggregation pathway (Figure 4). Native state
aggregation shifts the equilibrium towards the native state,
increasing ΔG at higher protein concentrations (Figure 5).
Aggregation from the denatured state has the opposite effect,
shifting equilibrium away from the native state and lowering ΔG
at higher protein concentrations. Hunky measures aggregation
propensity and pathway with either two concentration points
for an Agg Path measurement, or over a multi-point concentration series for ΔGtrend.
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