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.
Challenges of Formulations
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.
Formulation Screen of
Trastuzumab using the SUPR-DSF
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.
The SUPR-DSF from Protein Stable demonstrates high data quality, capable of quantifying multi-domain unfolding events of the therapeutic antibody Infliximab during a formulation screen in a single 2-hour experiment. In this formulation experiment, we determined the optimal formulation for Infliximab to be a salt-based buffer with a pH of 7.2. This formulation improved the stability of Infliximab by increasing the melting temperature of the first transition by a 2.3°C and the second transition by 1.3°C.
Measuring Aggregation Propensity of a Protein using SUPR-CM
In this application note, the SUPR-CM fluorescence plate reader was used to determine the aggregation propensity of the model protein lysozyme in two different buffer solutions in order to illustrate how ICD can be used to assess which buffer condition would result in fewer aggregates.
Comparing pH and Buffer Solutions for
Stabilising a Monoclonal Antibody using the SUPR-CM High-Performance Plate Reader
Chemical denaturation experiments were performed in this study, on an IgG1 mAb to establish which buffer and pH improved the stability of the mAb the most. Equilibrated samples were prepared in 384-well microplates and rapidly measured with the SUPR-CM. Both MOPS and TRIS improved the C m values over phosphate and citrate. Quantification of more detailed denaturation data, obtained with SUPR-CM, revealed MOPS to have the higher C m1 value and higher ∆G° values. The high quality, detailed denaturation data produced with the SUPR-CM highlights the overall improvement in stability of the mAb when using a MOPS buffer at pH 7.25.
Statistical Repeatability of tm Values Highlights Utility of Protein Stability Screening Automation
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.
