Unchained Labs eBook - 12

Unleash your biologic * Protein characterization turned up to 11

SLS at two different wavelengths. An increase in the signal at
266 nm is usually picked up first, as it should be more sensitive
to changes in scattering intensity and discerns smaller particles.
Scattering intensity measurements at 473 nm are useful for detection of larger aggregates, and to differentiate samples when
the intensity has saturated at the lower wavelength. In this case
the mAb showed a sudden onset of aggregation (Tagg) at 78.2
°C, very soon after the second melting transition. Obtaining both
of these measurements at the same time makes it easier to rank
constructs or formulations, or screen excipients that prevent or
delay the onset of aggregation.

Sizing and polydispersity adds more

Figure 2.
Size and polydispersity for four different monoclonal antibodies after
storage at 4 °C. mAbs 1, 2, and 4 showed the expected hydrodynamic
diameter, and had polydispersity index values below 0.25, indicating
monodisperse populations. In contrast, mAb3 exhibited a diameter of
175.7 nm and a polydispersity index that suggested a high level of
aggregation in the sample.

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In addition to the Tm and Tagg values, DLS measurements can
provide valuable information about the size and polydispersity of
the molecules. Without adding any additional sample or transfer steps, these measurements can be collected on Uncle at the
beginning and end of a thermal ramp. This data can serve as an
assessment of sample quality, and to identify molecules that are
not monodisperse before they even undergo thermal stress.
Alternatively, DLS can provide more clarification for samples that
display unexpected behaviors in thermal ramp measurements.
In Figure 2, the size and polydispersity of four different monoclonal
antibodies were measured on Uncle at 0.5 mg/mL. After storage

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