• Document: Value of DSC in characterization and optimization of protein stability as compared to other thermal stability assays
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Value of DSC in characterization and optimization of protein stability as compared to other thermal stability assays 韩佩韦 Malvern Instruments 2016.02.28 Complex task of characterization and optimization of protein stability › Chemical stress (pH, ionic strength, etc.) › Physical stress (filtration, temperature, air-liquid interface) › Freeze-thaw-induced stress Challenge: numerous causes of protein instability matched by numerous remedies to be tested empirically  Buffering agents Stabilization might be  Amino acids achieved through :  Osmolytes • changes in bulk  Sugars and solutions properties carbohydrates  Proteins and polymers • specific interaction with  Salts protein  Surfactants • inhibition of a  Ligands... degradation pathway Profiling and optimization of protein stability remains a combination of trial-and-error and rational approaches 3 70 Understanding of factors (intrinsic and extrinsic) critical Buffer attribute 2 60 2 to protein stability on the molecular 50 level is needed for implementation of the rational approach 40 to optimization of protein stability. 1 30 20 10 0 10 20 30 40 50 Buffer attribute 1 Multiple biophysical methods used for characterization of protein stability › Mass spectrometry (MS) › Circular dichroism (CD) › Fourier transform infrared spectroscopy (FTIR) › Raman spectroscopy › X-ray crystallography › Nuclear magnetic resonance (NMR) › Near-UV CD › SEC › HPLC › Fluorescence › Static and dynamic light scattering (SLS and DLS) › Differential scanning calorimetry (DSC) › Analytical ultracentrifugation (AUC) Thermal stability as generic indicator of protein stability Long-term Stability Indicator: Existing solutions: costly stability Protein Assessment studies of long-term and multiple stability optimization Issues: samples. often of protein Involve lengthy incubation correlates stability times & many with thermal through analysis rounds stability studies of for multiple samples. thermal stability Existing ways to assess thermal stability: Subject protein solution to a temperature up-scan and monitor changes in a property DSC IF DSF UV DLS&SLS CD BIOCHEM. / Potency ASSAY Rheology What are we actually measuring? Instrumental Macroscopic average readout property • 1st principle physical relations • assumptions • simplification Molecular level Property, process DSC instrument readout is directly related to the thermal unfolding event Differential Scanning Calorimetry DSC • Directly monitors thermal unfolding event by measuring changes in apparent excess heat capacity of protein

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