Unchained Labs eBook - 11

Unleash your biologic * Protein characterization turned up to 11

The Uncle platform from Unchained Labs combines three different detection modes on a single instrument. Fluorescence,
static light scattering (SLS), and dynamic light scattering
(DLS) are used to characterize protein stability. Temperature
can be precisely controlled from 15-95 °C, and low (9 µL)
volumes of up to 48 samples can be run at once, so that
many of the key stability measurements can be made earlier.
This streamlines and simplifies data collection and analysis.

Thermal melting and aggregation onset
To determine the melting temperature of a protein, Uncle
measures the intrinsic fluorescence of tryptophan and
tyrosine residues as the protein undergoes conformational
changes. In most cases there is a change in the fluorescence
intensity or a shift in the peak as the protein unfolds due to
thermal stress. In Figure 1, a monoclonal therapeutic
antibody (mAb) was prepared at 0.5 mg/mL, and run with
a thermal ramp from 15 to 90 °C with a ramp rate of 0.3 °C/
minute. Two distinct transitions were detected by the
analysis software, with thermal melting points (Tm's) at
67.8 °C and 76.5 °C.
Aggregation is often linked to unfolding of proteins. Rather
than performing separate experiments to detect aggregation
in a different instrument, Uncle simultaneously measures
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Figure 1.
Melting temperatures and onset of aggregation for a monoclonal antibody
formulated at 0.5 mg/mL. The conformational changes of the protein are
analyzed by plotting the barycentric mean of the fluorescence signal over
the thermal ramp. Two distinct transitions are detected, as indicated by the
dashed green lines. Aggregation onset, indicated by the dotted red line, is
determined by measuring the increase in SLS intensity at 266 nm.

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