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9
APPLICATION NOTE
predictive models using DLS-derived k
D
values or
SLS-derived B
22
values measured at low (<20 mg/ml)
concentrations have been shown to be only moderately
good predictors of high-concentration behavior and
aggregation propensity.
Deviation from linearity using static light scattering
or absolute scattering intensity is a reliable method
for measuring high-concentration behavior, as it does
a direct measurement with a highly concentrated
sample. The ability to use low sample volume for this
measurement is crucial in early development stages,
when candidate sample is precious and scarce.
Here we have shown that the EAP (prototype)
Prometheus Panta gives k
D
values that agree well with
previous data. Furthermore, while the SLS capabilities
of the EAP Panta were not fully developed, the raw
intensity scattering from the instrumentation allows
for measurements parallel to the SLS deviation from
linearity. These results also resulted in the same
ranking selections as other instruments in the field.
Additionally, the Panta demonstrated exemplary
repeatability between measurements, and therefore
lower standard deviations between replicates.
Both the DLS and impending SLS capabilities of the
Panta showcase the importance of a full biophysical
profile of candidate molecules, and exhibit how low-
volume, low-concentration measurements can be used
to predict behaviour in higher concentration systems.
This is a benefit to researchers, especially those in early
development, who are o en working with limited sample
quantities for all of their assays.
An added benefit of the Prometheus Panta system
not addressed here is its ability to measure thermal
denaturation and turbidity. Furthermore, DLS
measurements can be run in parallel along the same
temperature denaturation ramp, giving additional
stability parameters such as T
size
(the temperature at
which a protein begins to increase in size) and T
scattering
(the
temperature at which scattering intensity, and therefore
size, begins to increase.) The Panta gave additional
insight that most of the candidate molecules do not
show a major increase in turbidity, but there is a
given temperature at which T
size
begins to increase,
indicating soluble aggregation (data not shown).
Merck Healthcare KGaA Darmstadt used the EAP
Prometheus Panta to make precise measurements
on their biologic samples. The information obtained
from the Panta allowed selection of an ideal candidate,
as well as the ideal buffer formulation to work in with
their candidates. These results show that the Prometheus
Panta gives exemplary, reproducible data that
corroborates previously obtained ranking parameters.
