9
APPLICATION NOTE
©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved.
were filled automatically from MTPs using the
NT.Robotic Autosampler. Filled capillary chips
were then automatically transferred to the
Prometheus NT.Plex and chemical denaturation
was measured. Chemical denaturation data were
fitted unattended by a three-state unfolding model
to yield ∆G°
app
. ∆G°
app
values were plotted versus
protein concentration to identify trends in ∆G°
app
.
All ∆G° values presented in the manuscript are
∆G°
app
values.
For nanoDSF measurements, mAb1 was diluted
into the respective formulations to reach a final
concentration of 5 mg/mL, and subsequently filled
into Prometheus Standard capillaries. Thermal
unfolding and aggregation was monitored in a
temperature ramp with 1 °C/min from 20 °C to 95 °C
with a resolution of ~ 20 data points/min. Analysis
of unfolding and aggregation was performed using
the PR.Control So ware.
For long term stability measurements, mAb1 was
stored at a concentration of 20 mg/mL at 5 °C
and 25 °C, respectively. HPSEC was performed
using a TSK Gel G3000 SWXL 7.5 mm x 300 mm
column (Tosoh, Bioscience, Tokyo, Japan) on a
Waters 22675 Alliance HPLC system connected
with an absorbance detector Waters 2487 (Milford,
MA, USA). Turbidity was measured in formazine
nephelometric units (FNU) using photometry of 90°
scattered light at 400-600 nm (2100 AN Laboratory
Turbidimeter, Hach, Loveland, CO, USA).
Acknowledgements
The authors thank Torsten Schultz-Fademrecht,
Anastasia Pusik and Andrea Eiperle for excellent
technical assistance.