Application Notes

Getting the full picture: predicting the aggregation propensity of mAbs using chemical and thermal denaturation on a single, fully automated platform

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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.

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