Application Notes

©2021 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. 2 APPLICATION NOTE With Prometheus Panta, it is now possible to do dynamic light scattering (DLS) experiments in tandem with nano-differential scanning fluorimetry (nanoDSF) and backreflection along an entire thermal ramp in order to gain conformational, thermal stability, and turbidity information about your formulation 1 . Prometheus's nanoDSF and turbidity measurements obtained from the NT.Plex instrument have been validated as industry-standard, but with the addition of DLS capabilities, it is crucial to ensure the same quality and reliability can be found in the Panta instrumentation. Merck's Protein Sciences group recently used the Prometheus Panta to evaluate the properties of a series of monoclonal antibody mutants with the objective of supplying data to machine learning programs to improve the candidate selection process 2 . DLS, nanoDSF, and backreflection were used to evaluate sequence mutations and determine how they affected molecule stability. It was crucial not only to evaluate these candidates with the added DLS ability, but also to determine whether the Panta could be integrated into their workflow without a loss in measurement accuracy from the nanoDSF and turbidity measurements they are typically performing. Once the data were collected in the Panta, they compared data for the same molecules measured previously on the Prometheus NT.Plex to ensure repoducibility. Methods Prometheus Panta measurements Antibody samples were purified to 1 mg/ml in 20 mM sodium acetate pH 5.5 buffer. Single replicates of each sample were run in the Early Access Program (EAP) Prometheus Panta with the 48 individual capillary adapter in place. UV LED excitation was 12% for nanoDSF and turbidity measurements; excitation was set to 100% for DLS experiments. Isothermal measurements for DLS were collected at 25 o C using the high-sensitivity mode prior to the thermal denaturation experiments. Temperature denaturation with nanoDSF, backreflection, and DLS acquisition was run from 25- 95 o C at 1 o C/min. Data was collected using a beta version of Panta.Control so ware, and a er converted to a format to allow analysis using Panta.Analysis so ware v1.1. Prometheus NT.Plex measurements Single replicates of each sample were run in the Prometheus NT.Plex. UV LED excitation was 40% for nanoDSF and turbidity measurements. Isothermal measurements for DLS were collected at 25 o C using the high-sensitivity mode prior to the thermal denaturation experiments. Temperature denaturation with nanoDSF and backreflection acquisition was run from 25-95 o C at 1 o C/min. Data was collected using version 2.2 of Therm. Control so ware.

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