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 Amino acids Osmolytes Sugars and carbohydrates Proteins and polymers Salts Surfactants Ligands... Stabilization might be achieved through : changes in bulk solutions properties specific interaction with protein inhibition of a degradation pathway
Buffer attribute 2 Profiling and optimization of protein stability remains a combination of trial-and-error and rational approaches 3 2 1 70 Understanding of factors (intrinsic 60 and extrinsic) critical to protein stability on the molecular 50 level is needed for implementation of the rational approach 40 to optimization of protein stability. 30 20 0 10 20 30 40 50 Buffer attribute 1 10
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)
Issues: Stability Indicator: Existing solutions: Thermal stability as generic indicator of protein stability Long-term costly stability studies of multiple samples. Involve lengthy incubation times & many analysis rounds for multiple samples. Protein long-term stability often correlates with thermal stability Assessment and optimization of protein stability through studies of 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. ASSAY Rheology / Potency
What are we actually measuring? Instrumental readout Macroscopic average property 1st principle physical relations assumptions simplification Property, process Molecular level
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 sample during temperature up-scan Circular Dichroism Spectropolarimetry In a limited set of buffers follows unfolding through changes of CD spectrum sensitive to secondary and tertiary structure of the protein. Light scattering techniques: DLS and DSLS Indirectly monitor thermal unfolding of proteins through changes in protein size and extent of protein aggregation Differential Scanning Fluorescence, DSF Indirectly monitors thermal unfolding of proteins through changes in hydrophobic area or content of beta-sheet structure accessible to environment-sensitive fluorescent dye Fluorimetery, intrinsic fluorescence Indirectly and locally follows thermal unfolding of proteins by monitoring changes in intrinsic fluorescence of tryptophan and tyrosine residues
Differential Scanning Calorimetry, DSC Thermal stability of proteins at different conditions (buffers, excipients, adjuvants, ligands) Temperature is ramped Thermal denaturation of protein is monitored
MicroCal VP-Capillary DSC System m 130 µl cell volume
DSC offers universal thermal stability assay Heat as universal readout directly related to protein unfolding event No optical artifacts, works in turbid solutions Works under controlled conditions in most of buffers Allows to study protein samples as they are Minimum assay development Delivers multiple descriptors of protein stability
Protein unfolding monitored in DSC
Protein unfolding monitored in DSC
Protein unfolding monitored in DSC
Protein unfolding monitored in DSC
Protein unfolding monitored in DSC
Protein unfolding monitored in DSC
Protein unfolding monitored in DSC
Protein unfolding monitored in DSC
DSC thermogram provides multiple descriptors of protein thermal stability T m - thermal transition midpoint, multiple T m for multiple domains T onset - onset of unfolding DT 1/2 - width of a peak at half maximum hight DH cal, DH vh calorimetric and van t Hoff enthalpy Assessment of oligomerization and aggregation propensity Percent reversibility of the transition Kinetics of irreversible process DC p for reversible unfolding
Insights on protein stability accessible with DSC Shifts in Tm values ( C): Higher temperature = more stable Lower temperature = less stable Shifts in Tonset values ( C): Higher temperature = more stable Lower temperature = less stable Lowering of DH, area under the curve: decrease in the content of folded protein DH cal /DH vh ratio Measure of the size of the cooperative unit Changes in T 1/2 values (transition width, C): Smaller width = more cooperative unfolding usually associated with a compact structure Larger width less cooperative usually associated with a relaxed, partially unfolded structure Courtesy of Dr. Katherine E. Bowers, FUJIFILM Diosynth Biotechnologies U.S.A., Inc.
C p (kj K -1 mol -1 ) Use Tm to compare native, altered and mutant forms Native Mutant Phosphorylated Complexed Temperature ( C)
Tm as stability indicating parameter. Case study 1. DSC sensitive to changes in stability arising from a common chemical degradation pathway For each of the studied proteins (2 mabs and 2 fusion proteins), the melting temperature (Tm) decreased linearly as a function of oxidation. For one protein, DSC was shown to be a leading indicator of decreased antigen binding suggesting a subtle conformation change may be underway that can be detected using DSC prior to any observable impact on product potency. Arthur, K. K., Dinh, N. and Gabrielson, J. P. (2015), Technical Decision Making with Higher Order Structure Data: Utilization of Differential Scanning Calorimetry to Elucidate Critical Protein Structural Changes Resulting from Oxidation. J. Pharm. Sci., 104: 1548 1554. doi: 10.1002/jps.24313
Different T m at different protein concentrations indicative of protein oligomerization/ aggregation propensity Monomeric or not? T m shifts detect oligomers T m may shift with sample concentration Monomer Tm is concentration independent Monomeric protein Oligomeric protein
Cp (kcal/mole/ o C) Multiple Tms. DSC allows to address protein stability on domain level. Case study 2. Protein engineering guided by DSC. 150 Minor differences in primary sequence can have a big impact on antibody stability Stability of each domain can be assessed The least stable Fab variant expressed poorly and quickly formed high MW aggregates. Most stable antibody construct identified based on Tm of the Fab domain. 100 50 0 T m 2 = 73.3 C T m 1 = 60.4 C T m 3 = 82.0 C 30 40 50 60 70 80 90 Temperature ( o C) Demarest et al, Malvern Application note
Cp (kcal/mole/ o C) Tonset. Shift in onset of thermal unfolding correlates with protein stability and aggregation propensity. Case study 3. Cp (kcal/mole/ o C) 10 5 0 Data: BpaseTBSLa_cp Model: MN2State Chi^2 = 8813.30 T m 1 17.85 ±0.1932 DH1 2.19E3 ±181 DH v 1 1.13E5 ±1.06E4 T m 2 30.40 ±0.6202 DH2 1.41E4 ±2.8E3 DH v 2 8.04E4 ±4.94E3 T m 3 34.43 ±0.0458 DH3 4.01E4 ±2.68E3 DH v 3 1.25E5 ±2.92E3 onset at 12ºC BPase 8.3uM=0.23mg/ml BB293 in 20mM TBS + 200mM L-alanine ph 8.0 Protein X Construct 1 0 10 20 30 40 50 Temperature ( o C) Tm=34.5ºC DSC: 10ºC upward shift of the lowtemperature transition stabilizes Protein X kinetically. 5 4 3 2 1 0-1 -2 onset at 22ºC Protein X Construct 2 Tm=40ºC ca 0.22 mg/ml BPase BB384 in 20 mm TBS, ph 8.0 0.3% DMSO Data: BB384TBS05_cp Model: MN2State Chi^2 = 7482.82 T m 1 23.73 ±0.0840 DH1 877 ±93.6 DH v 1 4E5 ±5.32E4 T m 2 34.30 ±0.1938 DH2 1.14E4 ±932 DH v 2 9.88E4 ±4.06E3 T m 3 40.10 ±0.0854 DH3 2.64E4 ±939 DH v 3 1E5 ±2.15E3 20 30 40 50 60 Temperature ( o C) Construct 1 Construct 2 SEC SEC: Protein X homogeneity and stability to aggregation has dramatically increased
T 1/2 and Tm. Multiple metrics of protein thermal stability make buffer optimization /preformulation funnel more efficient. Case study 4. 12 selected Rank by T m 19 formulations tested Focus with T 1/2 5 focused on Katherine E. Bowers, Malvern Application Note
DH cal. 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. Malvern Application Note
DH cal /DH vh ratio as indicator of size of cooperative unit for thermal unfolding DH vh = DH cal cooperative unit and molecular weight are the same: largely reversible unfolding of one single domain DH vh < DH cal - cooperative unit is smaller than molecular weight: intermediates DH vh > DH cal cooperative unit is bigger than the molecular weight: oligomers or overestimated concentration of folded protein
Cp (cal/ o C) Ratio of calorimetric to van t Hoff enthalpy DH/DH vh, can be indicative of protein oligomerization state. Case study 7. 400 350 300 250 200 150 100 Data: Data2_cp Model: MN2State Chi^2/DoF = 99.35 T m 69.15 ±0.021 DH 3376 ±17.2 DH v 1.014E54 ±640 mgst2 50 0-50 20 40 60 80 100 Temperature (Deg. C) Ratio of van t Hoff to calorimetric enthalpy 3 indicates that protein unfolds as an oligomer (possibly trimer).
Existing ways to assess thermal stability: Can DSC make a difference? DSC IF DSF UV DLS&SLS CD BIOCHEM. ASSAY Rheology / Potency
How DSC performs against other thermal stability assays? Case study 8. Buffer optimization conducted with DSC, DSF and IF. Thermal stability screening of >500 samples 3 different proteins (2 mabs and one receptor protein 30 different buffer conditions. Thermal unfolding of the protein was followed with DSC, DSF and IF.
Proteostat Tm C Sypro Orange Tm C When it works DSF correlates rather well with DSC (Tm1) Screens at: 0.3 mg/ml 80 75 70 65 60 55 50 45 80 75 70 65 60 55 50 55 60 65 70 75 80 R²= 0.8023 DSF R²= 0.7253 DSF 60 65 70 75 DSC Tm C
Proteostat Tm C Sypro Orange Tm C When it works DSF correlates rather well with DSC (Tm1) Screens at: 0.3 mg/ml 80 75 70 65 60 55 50 45 80 75 70 65 60 55 50 55 60 65 70 75 80 R²= 0.8023 BUT does DSF always work? DSF R²= 0.7253 DSF 60 65 70 75 DSC Tm C
DSF is not truly universal assay DSF is not applicable to all proteins and all buffer compositions. One out of 3 proteins in this study was not amenable to DSF with either of the extrinsic dyes SyproOrange or ENZO. Self-associating excipients corrupted the readout of DSF. Domain resolution was not achieved with DSF
DSF ProteoStat Enzo DSF Sypro Orange MicroCal Cap DSC automated Performance varies a lot between DSC and DSF techniques Technique Protein mab1 mab2 % samples amenable to analysis % samples with domain resolution % samples amenable to auto data analysis 100 100 100 Receptor X 100 one peak 100 mab1 91 28 66 mab2 74 50 30 Receptor X failed mab1 100 0 27 mab2 100 0 0 Receptor X failed
Domain resolution generally only achieved in DSC Domain resolution for all buffers was only achieved with DSC. DSF is less effective when looking at domains and their stabilization. Peak amplitude in DSF is not domain-specific. Direct nature of DSC readout ensures robustness of baseline definition and domain resolution.
Why/when data on thermal stability of individual domains can be of importance Guiding protein engineering work Comparing proteins produced with different expressions systems (glycosylation effect on domain stability) Relating changes in functional activity to stability changes of individual domains Selecting optimal condition during pre/formulation studies Characterization of Higher Order Structure (HOS) Comparability analysis
DSC vs IF in the buffer screen of mab1 sample. IF data can be difficult to interpret IF readout susceptible to optical artefacts like quenching, innerfiltering, aggregation, and light scattering
DSC has non-optical readout. DSC measurements are not affected by sample turbidity or presence of self-associating reagents in the buffers. DSF DSC DSC can be applied to the analysis of samples from long-term and stress-stability studies DSC gives means to assess conformational equivalence/biocomparability of protein batches originating from different processes and sources
DSC readout is directly related to protein concentration and conformational state. DSC thermogram is a fingerprint for assessment of conformational equivalence and Higher Order Structure. Three lots manufactured at different sites DSC verifies no difference in stability and solubility between DP lots Lot 3 Lot 2 Lot 1 Reference Malvern Application Note
Summary DSC is truly universal and generic assay. DSC is the only thermal stability (TS) method with direct readout DSC is information rich (multiple descriptors of thermal unfolding) allowing for rational data interpretation for protein characterization and stability screening DSC thermograms are sample specific, quantitative and reproducible. Allows for similarity analysis. DSC is a well-established tool used to validate other TS assays.
With Differential Scanning Calorimetry you can: Characterize protein stability Characterize domain structures Assess oligomerization state Screen engineered and recombinant proteins for stability and manufacturability Determine optimum solution conditions for protein purification and formulation development Assess biocomparability and biosimilarity Study/screen protein-ligand interaction
Thank you for your attention! Questions?