Technical Notes

TECHNICAL NOTE 3 ©2020 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Lysozyme powder was resuspended in 25 mM histidine buffer pH 5 supplemented with 130 mM NaCl to a final concentration of 20 mg/mL. The solution was centrifuged at 16,000 g for 15 minutes at 4°C before the supernatant was filtered through a 0.2 µm syringe filter. Dilutions of 10, 5, 2.5, 1 and 0.5 mg/mL were then prepared for size analysis. NISTmAb was obtained at 10 mg/mL in 12.5 mM histidine buffer pH 6. A er centrifuging at 16,000 g for 15 minutes at 4 °C, it was further diluted to 8, 6, 4,2, 1, 0.5, 0.25, 0.1 and 0.05 mg/ mL using the formulation buffer. The r H for the concentrations >0.5 mg/mL for each protein was evaluated using a cumulant fit to the autocorrelation function; the r H for the concentrations ≤0.5 mg/mL of both proteins was evaluated using a size distribution model that typically performs better for concentrations at the lower limit of detection. Figure 1 shows the results of the cumulant fits for experiment. The r H values for Lysozyme do not vary, and agree well with the literature value of 1.9 nm 1 . The r H values for NIST mAb vary based on concentration; they in good agreement for published literature of 4.98 nm at standard 10 mg/mL concentration 2 . For details about how concentration affects r H , see the NanoTemper Technical Note regarding k d determination 3 . Even for protein concentrations down to the detection limits of the instrument, Prometheus Panta is able to reliably and consistently determine sample size 10 -1 10 0 10 1 0 1 2 3 4 5 6 NIST mAb Lysozyme Intra-assay precision Prometheus Panta is equipped with a sample tray that allows either up to 48 single capillaries — loaded individually onto the instruments — or 24 capillaries arranged on a chip for faster and more convenient sample loading. To determine the intra-assay precision of the DLS optical module across the entire capillary tray, independent of its format, 100 µg/mL NISTmAb diluted in 12.5 mM histidine buffer pH 6 was loaded into 48 single capillaries or into the capillary chip and measured. The same analysis was then done with 0.5 mg/mL Lysozyme in 25 mM histidine buffer pH 5 supplemented with 130 mM NaCl. The r H values were evaluated using a size distribution fit to the autocorrelation function. Figure 1: Sizing accuracy of Prometheus Panta's DLS optical module, determined for different concentrations of Lysozyme and NISTmAb (n = 5 independent measurements, error bars represent the standard deviation). The r H were evaluated using a cumulant fit to the autocorrelation function.

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