Technical Notes

Repeatability assessment of Tycho NT.6 for better protein quality checks

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2 TECHNICAL NOTE ©2017 NanoTemper Technologies, Inc. South San Francisco, CA, USA. All Rights Reserved. Figure 1: High repeatability of data generated on the Tycho NT.6 system A) Overlay of 24 unfolding profiles of trastuzumab from duplicate measurements over a time period of 17 days. Two T i s of 74.7 °C and 86.2 °C were identified. B) The two inflection temperatures T i 1 and T i 2 for each measurement were automatically calculated by the Tycho NT.6 software. Error bars represent the standard deviation. The standard deviation of the results for each identified T i were less than 0.01 °C. A strong overlap of the 24 unfolding profiles was observed (Figure 1A). The high precision of the thermal unfolding experiments is illustrated by the negligible deviation of the separate T i values throughout the entire measurement period (Figure 1B), as well as by the very small relative standard deviation (RSD) of < 0.1 °C (Table 1). In addition, the day-to-day repeatability and consistency of the fluorescence detection was evaluated by analyzing the initial ratio values. The analysis yielded a standard deviation (SD) < 0.001, demonstrating the exceptional precision of the Tycho NT.6 system optics. The relative standard deviation of the initial fluorescence ratio was 0.11 %. Antibodies are an invaluable detection tool used by most researchers working with proteins. Scientists can purchase antibodies from numerous commercial sources as well as generate their own. While validation is provided with commercial reagents including antibodies, o en the quality and functionality of the product is poorly controlled and researchers are le to deal with the lack of specificity or modify their experimental plans. On top of this, mishandling and improper storage of antibodies can result in denaturation or reduced specificity which translates into irreproducible results or incorrect conclusions. Tycho NT.6 provides a rapid and precise way to monitor protein quality within minutes. Requiring only tiny amounts of sample, the thermal stability of any protein sample is swi ly determined. We demonstrate, using trastuzumab as reference antibody sample, the robust repeatability of multiple measurements generated by the system. Trastuzumab at 2 mg/ml in PBS buffer was repeatedly measured in duplicate over a period of 17 days, storing the sample at 4 °C between measurements. In total, 24 unfolding profiles were recorded (Figure 1A). The repeatability of the two inflection temperatures (T i ), or the points on the curve which represent unfolding of the sample due to thermal treatment, was recorded. In addition, the initial fluorescence prior to thermal treatment of the sample was measured in each experiment.(Figure 1B, and Table 1).

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