DSF Application Notes

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PP038 Application Note Amends V2 1600 X 1000

IDENTIFICATION OF AAV SEROTYPES AND CHARACTERISATION OF THEIR STABILITY

Measuring the thermal stability of three AAV serotypes, AAV2, AAV5, and AAV6, using the easy plate reader technology for protein stability screen: the SUPR-DSF. The SUPR-DSF measured the intrinsic fluorescence of each serotype during a thermal ramp to generate melt curves. In addition, we performed a concentration dilution series on AAV2 and AAV6 to determine the minimal sample requirements. All samples were performed at a well volume of 10μL in triplicate on a single 384-well microplate, emphasising the high-throughput possibilities of the instrument.

PP038 Application Note Amends PSL Resized 3 1

Statistical Repeatability of tm Values Highlights Utility of Protein Stability Screening Automation

Analysing the statistical distribution of over 6000 repeat lysozyme samples gave accurate T_m 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.

PP038 Application Note Amends PSL Resized 2 Image11 Tris Naci Concentration Stabilisation 01

Targeting Optimal Buffers for Downstream Crystallisation Screening

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.

PP059 Application Note 1000X1600 Thumbnail 08 Formo Screen Infliximab Top Stabiliser 01

Finding Formulations Faster with SUPR-DSF

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.

PP038 Application Note Amends PSL Resized 2 Image09 SUPR DSF Vs DSC Nistmab Comparison 01

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.

PP038 Application Note Amends PSL Resized 2 Image01 Fraction Unfolded All Stabilisers 01

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.

PP038 Application Note Amends PSL Resized 2 Image04 Comparison Between Wild Type And Mutants 01

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.

PP038 Application Note Amends PSL Resized 2 Image05 Midpoint Of Infections 01

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.

PP038 Application Note Amends PSL Resized 2 Image02 Gibbs Free Energy Against Lysozyme Concentration 01

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.

PP038 Application Note Amends PSL Resized 2 Image03 Buffer Comparison 01

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.

PP038 Application Note Amends PSL Resized 2 Image06 Peroxide Degradation 01

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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