Unchained Labs eBook - 18

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

A

The analysis of chemical denaturation curves produces the two parameters,
C1/2 and m, needed to calculate the native state ΔG by ΔG = C1/2*m.1 C1/2 is the
denaturant concentration at the midpoint of the unfolding transition, which
represents the state in which half of the protein population is unfolded. ΔG
and C1/2 report on the structural stability of proteins, where higher ΔG (or C 1/2)
indicates a more stable protein. Hunky measures C1/2 with 8 to 12 point
denaturation curves, reducing experimental time and sample requirements.
ΔG is measured with 24 to 48 point denaturation curves to quantify stability
and determine the fraction of unfolded protein present.
ΔG can provide higher resolution between similar molecules or sample conditions
that are often indistinguishable by qualitative stability measurements like Tm.
Tm and ΔG were measured for Adalimumab in 10 mM acetate pH 4.0, 4.3, and
4.6. Adalimumab underwent a single unfolding transition in all 3 formulations
over a 20-95 °C temperature ramp. The Tm values were highly similar for the
three formulations making it difficult to identify the most stable conditions
(Figure 3A). To clarify the stability ranking, ΔG was measured on Hunky. All
Adalimumab formulations underwent two distinct unfolding transitions when
denatured with 10 M Urea (Figure 3B). The difference in ΔG(1) for 10 mM
acetate pH 4.0 and pH 4.6 corresponds to the contribution of 72 ppm or 0.05
ppm of denatured protein, respectively. ΔG identified 10 mM acetate pH 4.6
as the most stable formulation studied and filled in the blanks left by Tm.

B

Figure 3. The Tm of Adalimumab over a small pH range does
not yield a stability ranking (A), but ΔG measurements make
it possible to differentiate formulations (B).

18

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Unchained Labs eBook

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