Application Notes

2 drug approval [1, 2]. The growing number of mAbs and mAb variants in development pipelines calls for biophysical methods which can quickly assess those parameters [3, 4]. Screenings for conditions and antibody constructs in early stages of development aim to identify most promising candidates to meet regulatory requirements for drug approval. Here, we use a small scale formulation screen for a therapeutic monoclonal IgG1 antibody to demonstrate the capability of the Prometheus NT.48 in determining critical stability parameters. One approach to determine the conformational stability of proteins is to follow their unfolding in temperature gradients [5-9]. Increasing the temperature of the sample results in a transition of proteins from the folded to the unfolded state (Figure 1C). The temperature at which this transition occurs, the T m , is used as a surrogate parameter for the thermal stability of the protein. The Prometheus NT.48 uses nanoDSF to assess mAb conformational stability in thermal gradients by following changes in tryptophan fluorescence emission [8, 9]. Owed to the high sensitivity of the fluorescence detection in capillaries, even minute changes in antibody conformation can be detected, allowing for the unambiguous identification of multiple unfolding events which can be attributed to the different mAb subdomains. At the same time, the Prometheus NT.48 can detect changes in colloidal stability and temperature-induced aggregation. This is achieved by detection of the backreflection intensity of a light beam that passes the sample twice (Figure 2). Figure 2: Schematic representation of the backreflection principle to detect protein aggregation. (Left) light passes the capillary, is backreflected into the detector and light intensity is quantified. (Right) particles scatter light, leading to an extinction of the incident light and thus a reduction of backreflected light, which is a direct measure for protein aggregation. Upon aggregation, the intensity of the backreflected light decreases due to light scattering, and thus serves as a measure for total aggregation in a sample. Importantly, backreflection and fluorescence are detected simultaneously, allowing for maximal scanning speed and data point density. A simultaneous assessment of conformational and colloidal stability is a powerful approach to predict long term stability. Every antibody – irrespective of its conformational stability – is in equilibrium between native (folded) and unfolded conformational states (Figure 1A). If the unfolded state of the antibody shows a strong tendency to form aggregates, it precipitates out of solution, resulting in more antibody becoming unfolded driven by the law of mass action (Figure 1B). Thus, strong aggregation of unfolded antibodies has to be prevented, e.g. by identifying optimal formulation compositions or molecular modifications that prevent aggregation. Simultaneous backreflection and fluorescence analysis using the Prometheus NT.48 provides several important information: Firstly, it is possible to directly correlate thermal and colloidal stability, meaning that aggregation-causing unfolding events can be identified, and more importantly, aggregation onset temperatures can be determined. Secondly, the overall degree of aggregation of the unfolded state of an antibody can be determined, which can vary significantly between different formulations and antibody types. Results We tested the thermal stability of an IgG1 antibody at concentrations of 1 mg/ml in acetate buffer with pH values between 4 and 6.5, and in presence and absence of 130 mM NaCl. For this, 10 µl of each sample were loaded into standard-treated Prometheus NT.48 capillaries and subjected to a thermal ramp from 40 °C to 95 °C at a heating rate of 1 °C/min. The resulting unfolding signals in the F350/F330 ratio showed two clearly defined unfolding transitions (Figure 3A). A decrease in pH resulted in a shift of the unfolding transition midpoints to lower temperatures (Figure 3A and 4A). This effect was even more pronounced in presence of 130 mM NaCl, showing that the addition of salt leads to a conformational destabilization of the antibody. In

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