Once candidates have passed through early development and optimisation stages, it is important to understand how manufacturing, storage, transport and delivery processing steps impact product integrity and safe lifetimes. The goal is to form a suitable buffer that confers the best conditions in which the protein molecule is most stable for these processes. Testing, of course is best performed in real-time, however, when the ideal scenario includes testing for stability over several years, more predictive analyses become necessary. Testing multiple formulations under a range of conditions helps rank order the effects and potentially aids understanding and prediction of long-term stability.
There are many buffers and excipients available to choose from the GRAS (Generally Regarded As Safe) list, which offers a wealth of combinations, although most scientists will have a starting point in mind. Choosing one or two types of buffer, perhaps at different concentrations and pH and then adding a few excipients also at a range of concentrations soon adds up to a significant amount of work preparing solutions. The selected molecule(s) is then introduced at various concentrations, sometimes even up to therapeutic levels. Using automated liquid handling becomes essential with this amount of work.
The SUPR-DSF is the perfect tool for rank ordering of thermal melting points – an indicator of protein stability – on such a large scale. Reading directly in the 384-well microplates, the formulations can be prepared in, increases workflow efficiency, reduces costs and errors from switching to alternative measuring methods and delivers fast and accurate results. The connectivity of microplates cannot be underestimated when working with these many variations. Further, the SUPR-DSF can be easily accessed by robotic plate handlers, thus facilitating automation if required, and with its unique design and epifluorescent detection, is not affected by higher sample concentrations such as those at therapeutic levels. With the option of running the SUPR-CM side by side, a system designed for chemical melts of the formulated samples, orthogonal data gives deep insight into the stability profiles.
Scatter plot of 96 different formulations of Infliximab. With two transitions and, therefore, two melting temperatures, the best conditions conferring stability can easily be identified. Samples were run in triplicate in a 384-well plate in less than 2 hours.
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.
The use of the SUPR-DSF to study binding interactions has demonstrated that this technique offers a viable and unique solution. The SUPR-DSF offers an intrinsic fluorescence-based high-throughput methodology that is free in solution, with all measurements carried out directly in the 384-well plate-based format. The SUPR-DSF rapidly provided highly precise data with excellent sensitivity in the study of Carbonic Anhydrase with TFMSA. The values obtained agree with previously published literature values for KD and prove that the SUPR-DSF can be used as either a pre-screening or conformational tool for ligand binding studies.
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.
