4
Formulations were reconstituted with MilliQ
water, and a lysozyme stock solution (5 mg/ml
in MilliQ water) was added to formulations to
reach a final concentration of 1 mg/ml
lysozyme. Thermal unfolding of lysozyme in
the different formulations was analyzed using
the Prometheus NT.48. Briefly, 10 µL of each
of the 25 formulations were aspirated into
standard-treated capillaries by capillary action
and analyzed using the Prometheus NT.48 at
a scan rate of 2
°
C/min from 20 °C to 95
°
C.
The software analyzed the thermal
denaturation curve and determined the onset
(T
onset
) and unfolding transition temperatures
(T
m
) of each of the 25 formulations.
The data were then analyzed using DOE
software, and Pareto, response-surface, and
interaction plots were generated to define the
formulation design space. An optimum
formulation with the maximum T
m
was
identified.
Results and Discussion
DSF profiles of iFormulate™ formulations.
Figure 2 shows thermal unfolding curves for
the 25 iFormulate™ formulations which differ
in pH, ionic strength, trehalose concentration,
and buffer concentration. Depending on the
formulation composition, the T
m
varied
between 70.3
°
C and 78.1
°
C. Figure 3 shows
the 1
st
derivative analysis of the nanoDSF
profiles to better illustrate T
m
shifts between
the formulations. The data generated by the
Prometheus NT.48 are highly precise and
reproducible as shown in Figure 4 and
Table 1. The color-coded profiles are the five
replicate formulations in the iFormulate™
design that are placed randomly in the plate.
The DOE analysis resulted in a residual S.D.
of 0.22 and replicate S.D. of 0.16, indicating
the high precision and reproducibility of
Prometheus NT.48 nanoDSF.
Figure 2: Prometheus NT.48 nanoDSF thermal unfolding profiles of iFormulate
TM
formulations of lysozyme. Thermal
unfolding was recorded at 2 °C/min from 20
°
C to 95
°
C, A total of 1.4 mg of protein was required for analyzing
28 experimental trials in 35 minutes. The y-axis is F350/330 fluorescence ratio and x-axis is temperature (
°
C).
