Application Notes

©2021 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. 2 APPLICATION NOTE and ultimately be used at these higher concentrations. Because of this, researchers must have a way to use low amounts of material to assess how their protein therapeutics will behave once they are at their clinical concentration. There are many parameters researchers must evaluate their candidates on prior to reaching pre-clinical testing stages 2 . Dynamic light scattering (DLS) and static light scattering (SLS) offer important information about size and dispersity of particles in solution. DLS allows researchers to measure the hydrodynamic radius of a particle as well as the distribution of particle sizes in a solution, and can be used to determine the self- interaction parameter k D of a protein. SLS measures the average molecular weight in solution and can be used to find the second virial coefficient B 22 of a candidate. Researchers aim to minimize the self- interaction of proteins in order to avoid aggregation and therefore effectiveness of candidates once they reach their scaled-up concentrations for the clinic. Together, these parameters inform researchers about their developmental candidates, and can be used to monitor self-interaction of a system and optimize it to avoid aggregation in the final scaled-up formulation. The Prometheus Panta is a DLS-enabled instrument that uses nano-differential scanning fluorimetry (nanoDSF), backreflection (for turbidity, or large amorphous aggregates), and DLS to enable researchers to measure their candidate's biophysical characteristics using minimal sample consumption 3 . Merck Darmstadt used the Panta to measure and rank the self-interaction parameters of several protein biologic candidates, and compared those rankings to that of other systems. The data obtained from Panta agreed with that from other systems, and demonstrated exemplary linearity of measurement. Methods Panta DLS Screening Five candidate molecules were prepared in several different buffer conditions, exhibiting a range of salt concentrations and pHs. Each molecule and buffer system was prepared at six concentrations, from 2.5 to 15 mg/ml. Triplicates of 10 ul each were run in the prototype early access program (EAP) Panta system on High Sensitivity mode. Values for k D s were determined using the beta (0.4) Panta Analysis so ware for EAP. Experiments were run in duplicates on the Prometheus Panta (NanoTemper Technologies, GmbH) and Wyatt DynaProII (Wyatt Technologies Europe, GmbH; denoted as "plate reader") device to compare the k D s for each candidate. Results were compared for both the agreement of the k D value obtained as well as the deviation between three replicates. Panta light-scattering experiments A bi-specific Fc-fusion was prepared in several buffer formulations and examined using the light scattering of the system in an EAP Panta. While EAP Panta was not yet calibrated for publication-standard SLS measurements, the scattering values obtained from the system could be used to provide rankings for SLS-deviation, similar to what was obtained from other systems.

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