Once a target of interest has been identified, the first stage of a large molecule discovery program is to find or create molecules of interest that have some level of effect on that target. Multiple ways exist, from the more “shotgun” phage display to a more targeted point mutation strategy, resulting in construct libraries of various sizes. Function is key – for example, does the molecule bind to the target? It does not matter how weak that binding may be, as that can be addressed downstream in an optimisation program. Another important factor is the molecule's stability, which indicates how likely the construct will move through the discovery pipeline and perhaps eventually become a marketed molecule. Having the most stable form of the protein, for example, an engineered antibody is a solid basis for further optimisation work before forwarding the candidates to the next development steps.
Often involving hundreds to thousands of early constructs, all of which have been produced in very small quantities, screening for stability requires higher throughputs, low sample usage and the ability to link seamlessly to other parts of the process, such as liquid handling and automation.
Using the SUPR-DSF from Protein Stable is the natural choice for construct stability screening with direct in plate measurements. This means that 384-well microplates can be used for sample preparation, and the plates can be directly processed in the SUPR-DSF. The rank order of stabilities can be produced in a matter of hours, not days and the data-rich spectra are used to predict anomalies such as self-association in addition to producing Tm.
Simple protein thermal melt analysis, ranking mutants against a wild-type. In this example, Mutant 14 destabilises and is not a good candidate. Mutant 01 increases stability the most and introduces a second transition to the melting profile. Mutant 08 is the strongest candidate with the largest increase in single transition Tm.
Analysing the statistical distribution of over 6000 repeat lysozyme samples gave accurate Tm values while providing a low standard deviation of only 0.14°C. The SUPR-DSF, therefore, has great utility within protein stability screening, connecting to supporting technologies, minimising handling errors and risk, and offering more samples with lower sample usage.
The stability of proteins in initial buffer conditions has been directly linked with crystal formation success in subsequent crystallography screens. One method of measuring protein stability in a buffer screen is with differential scanning fluorimetry (DSF). Comparison of thermal melting profiles of a protein in different buffers can indicate the conditions that increase protein stability. We show how the SUPR-DSF can be used to rapidly screen bovine Beta‑lactoglobulin (BLG) in a variety of buffer conditions to increase stability and subsequently aid in improving homogeneity in solution. The system’s 384-well plate format allows simple screening of commercially available buffer screens, plus replicates, in one thermal ramp experiment.
The NISTmAb is a widely characterised monoclonal antibody intended to be used as a reference molecule in the development of novel technology for therapeutic protein characterisation. In this application note, we use the SUPR-DSF to acquire differential scanning fluorimetry (DSF) data on the NISTmAb and compare the results against differential scanning calorimetry (DSC). The SUPR-DSF resolved all three domains of the NISTmAb: CH2 (69°C), CH3 (83°C), and the unusually high melting temperature of the Fab domain (94°C). In addition, the apparent melting temperatures agreed exceptionally well with the literature results. These results validate you can easily obtain high-quality protein stability information with the SUPR-DSF.
To illustrate the use of the SUPR-DSF for variant comparison and selection in early-stage discovery, we have used a model protein system to compare 16 analogues and identify the most stable ones for further processing.
In this study, we show the analysis of a thermal denaturation-based formulation screen of the commercially available therapeutic antibody Trastuzumab in 96 different conditions with the
SUPR-DSF instrument. Along with screening the stabilising agents, confidence in the results is gained as there is consistency with both Differential Scanning Calorimetry and the formulation used for the commercial drug Herceptin®. This screen was directly measured in a single 384-well microplate in less than 1.5 hours. This high throughput can be leveraged further through lab automation integration to screen thousands of samples per day.
