5
APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
sample were filled into 3 nanoDSF Grade Standard
Capillaries (NanoTemper Technologies), respectively,
and loaded into the instrument. mAb thermal
unfolding was monitored in a 1 °C/min thermal
ramp from 20 °C to 95 °C. T
m
values were determined
automatically by the PR.Control so ware.
µDSC thermal unfolding
Differential scanning calorimetry experiments were
performed on a MicroCal Auto VP-Capillary DSC
System (Malvern Instruments). For the measurements
about 400 µl sample was required.
To determine the background, reference and sample
cell were filled with formulation buffer and scanned
from 10 °C to 100 °C with a scan rate of 1 °C/min. To
determine the T
m
of the protein, the sample cell was
filled with the protein solution and the reference cell
with formulation buffer, and one heating scan from
10 °C to 100 °C at 1 °C/min was performed. A er the
protein scan, the cells were filled with 10 % Decon 90
solution and scanned with the same settings of the
protein sample. Subsequently, the cells were washed
extensively with highly purified water to remove the
detergent.
The background obtained from the corresponding
formulation buffer scan was subtracted from
the sample measurement. The T
m
values were
determined from the peak maxima of the unfolding
transitions a er baseline subtraction. Data analysis
was performed by using the system's Origin 7.0 DSC
so ware.
Material and Methods
Protein preparation
A therapeutic IgG1-type mAb was dialyzed into the
formulation buffers listed in Table 1. The protein
concentration was adjusted to ca. 1 mg /ml.
nanoDSF thermal unfolding
Thermal protein unfolding was monitored using
the Prometheus NT.48 instrument (NanoTemper
Technologies). For each buffer condition, 30 µl a
1 mg /ml mAb solution was prepared, and ~10 µl of
Table 1: List of formulation buffers.
Buffer substance
20 mM Acetate
20 mM Acetate
20 mM Acetate
20 mM Histidine
20 mM Histidine
20 mM Histidine
20 mM Phosphate
20 mM Phosphate
20 mM Histidine
20 mM Histidine
pH
3.0
4.0
5.0
5.0
6.0
7.0
7.0
8.0
6.0
6.0
Additive
0.02% PS20
0.02% PS80
