Value of DSC in characterization and optimization of protein stability as compared to other thermal stability assays

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00:00:00 Welcome
00:01:53 Complex task of characterization and optimization of protein stability
00:02:43 Challenge: numerous causes of protein instability matched by numerous remedies to be tested empirically
00:03:19 Profiling and optimization of protein stability remains a combination of trial-and-error and rational approaches
00:03:47 Multiple biophysical methods used for characterization of protein stability
00:04:14 Thermal stability as generic indicator of protein stability
00:05:22 Existing ways to assess thermal stability: Subject protein solution to a temperature up-scan and monitor changes in a property
00:05:51 What are we actually measuring?
00:06:52 DSC instrument readout is directly related to the thermal unfolding event
00:08:05 Differential Scanning Calorimetry, DSC
00:08:27 MicroCal VP-Capillary DSC System
00:08:44 DSC offers universal thermal stability assay
00:09:22 Protein Unfolding Monitored in DSC
00:09:44 Protein Unfolding Monitored in DSC
00:09:53 Protein Unfolding Monitored in DSC
00:10:01 Protein Unfolding Monitored in DSC
00:10:01 DSC thermogram provides multiple descriptors of protein thermal stability
00:10:50 Insights on protein stability accessible with DSC
00:12:08 Use Tm to compare native, altered and mutant forms
00:12:48 Tm as stability indicating parameter. Case study 1.
00:14:03 Different Tm at different protein concentrations indicative of protein oligomerization/ aggregation propensity
00:14:52 Multiple Tms. DSC allows to address protein stability on domain level. Case study 2.Protein engineering guided by DSC.
00:16:01 Tonset
00:17:03 T1/2 and Tm. Multiple metrics of protein thermal stability make buffer optimization /preformulation funnel more efficient. Case study 4.
00:18:28 DHcal. Area under DSC curve is an indicator of the content of folded protein material. DSC can be used to assess quality of recombinant proteins. Case study 5.
00:19:06 DHcal/DHvH ratio as indicator of size of cooperative unit for thermal unfolding
00:19:51 Ratio of calorimetric to van’t Hoff enthalpy DH/DHvH, can be indicative of protein oligomerization state. Case study 7.
00:20:37 Existing ways to assess thermal stability: Can DSC make a difference?
00:20:55 How DSC performs against other thermal stability assays? Case study 8.Buffer optimization conducted with DSC, DSF and IF.
00:21:39 When it works DSF correlates rather well with DSC (Tm1)Screens at: 0.3 mg/ml
00:22:14 When it works DSF correlates rather well with DSC (Tm1)Screens at: 0.3 mg/ml
00:22:19 DSF is not truly universal assay
00:22:49 Performance varies a lot between DSC and DSF techniques
00:24:09 Domain resolution generally only achieved in DSC
00:25:14 Why/when data on thermal stability of individual domains can be of importance
00:25:51 DSC vs IF in the buffer screen of mAb1 sample.
00:26:59 DSC has non-optical readout. DSC measurements are not affected by sample turbidity or presence of self-associating reagents in the buffers.
00:28:59 DSC studies protein samples as they are. No extrinsic or intrinsic probe required.Impact of buffer conditions on mAb1 thermal stability scored differently by DSC and DSF due to probe-related artefacts.
00:30:45 DSC Readout
00:31:35 DSC serves as golden standard tool for validation and interpretation of data from other thermal stability assays
00:31:58 DSC assesses feasibility of indirect thermal stability assay (DSF). Case study 9.
00:34:02 DSC confirmed stabilizing effects of fragment hits from DSF primary screen but uncovers a false negative. Case study 10.
00:35:16 Caveats of indirect thermal stability assays. Fluorescence probe lowers compound and/or protein solubility?
00:36:46 Summary
00:37:21 With DSC you can:
00:37:56 Thank you for your attention
00:40:07 Contact Information

Differential scanning calorimetry (DSC) is a well-established technique for biomolecular stability studies. The technique is based on forced thermal denaturation of the biomolecules. It is extensively used for characterization of protein stability, comparisons between native and mutant variants of proteins, for optimization of buffer conditions, and for confirmation of molecular interactions including quantitative determination of affinities. DSC is also one of the main thermal stability assays used in biopharmaceutical development for construct selection, manufacturability assessment, optimization of bioprocess, biocomparability analysis and pre/formulation studies.

The key benefit with DSC as compared to other thermal stability assays is that it is based on heat measurements and therefore allows for the direct characterization of thermal unfolding of biomolecules without the need for extrinsic or intrinsic fluorescence reporter. Furthermore, the lack of spectroscopic readings means that the samples do not have to be optically clear. In addition, the characterization is not limited to the unfolding transition temperature (Tm).

DSC provides multiple descriptors of unfolding transition which facilitates selection of the best construct/condition and allows to fingerprint higher order structure and to compare stability of individual domains. DSC can also provide data on the forces involved in folding of biomolecules and the mechanisms by which they unfold. This webinar presents results of the case studies demonstrating the advantages of DSC for characterization and optimization of protein stability, comparability analysis, elucidation of buffer-dependent protein oligomerization and unfolding mechanism. The benefits of DSC as compared to other thermal stability assays are discussed.