Differential Scanning Fluorimetry Application Notes


Application Note 01 – 15

Differential Scanning Fluorimetry Reaches New Peaks on the NISTmAb

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.

Application Note 01 – 4

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.

Application Note 01 – 10

Speeding Up Early Stage Biotherapeutic Discovery with Next Generation Differential Scanning Fluorimetry

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.

Application Note 01 – 11

Using High Throughput Differential Scanning Fluorimetry to Obtain Binding Parameters

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.

Application Note 01 – 5

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.

Application Note 01 – 8

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.

Application Note 01 – 13

Quantification of Monoclonal Antibody Stability Change After Forced Degradation Studies

Forced degradation studies are routinely employed during the development of new biologics. Along with providing evidence for the mechanisms by which a mAb can degrade, the change in stability mimics the observations when comparing different antibody constructs.

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