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High throughput protein stability analysis DSF system
NegotiableUpdate on 12/29
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Overview
Differential Scanning Fluorescence (DSF) is a fluorescence detection method based on protein thermal denaturation process in high-throughput protein stability analysis. DSF (Intrinsic Fluorescence Method) does not require any labeling of the test sample or the use of any fluorescent probes, and can quickly measure the entire sample microplate, providing high-throughput data to assist in the screening of sample candidates and formulation components.
Product Details
Differential Scanning Fluorescence (DSF) is a fluorescence detection method based on protein thermal denaturation process in high-throughput protein stability analysis.
Protein stability
Protein stability is one of the key quality attributes of biotechnology drugs, which determines the efficacy, production feasibility, safety, and shelf life of biological drugs. The stability of advanced protein structures ensures that proteins remain active and safe throughout their shelf life.
Stability studies are used to confirm the stability of high order structures (HOS) of proteins under different conditions, such as temperature, pH, excipient composition, and storage time, in order to determine appropriate storage and transportation conditions. Meanwhile, regulatory agencies such as FDA and EMA also require extensive stability data before authorizing the use of biologics for approval. Therefore, understanding and ensuring the advanced structural stability of therapeutic protein drugs is a necessary condition for biopharmaceutical companies to develop safe and effective drugs. Researchers need to analyze and evaluate the advanced structural stability of proteins at multiple stages of research and production.
Common analysis methods for protein stability
The stability analysis of proteins is usually studied using the following methods:
Biochemical methods
Circular Dichroism Spectroscopy (CD)
Differential Scanning Calorimetry (DSC)
Quantitative PCR (qPCR) technology (exogenous fluorescent DSF)
Dynamic Light Scattering (DLS) and Static Light Scattering (SLS) Methods
Differential Scanning Calorimetry (DSC) is often regarded as the most important solution for protein stability analysis. However, due to the rapid analysis requirements of a large number of samples stored on microporous plates (such as 96 or 384 well plates), the application of DSC in drug screening and early development is somewhat limited. Therefore, DSF (intrinsic fluorescence method) has become a popular and cost-effective solution.
DSF (Intrinsic Fluorescence Method) does not require any labeling of the test sample or the use of any fluorescent probes, and can quickly measure the entire sample microplate, providing high-throughput data to assist in the screening of sample candidates and formulation components.
The DSF method rapidly screens a large number of samples, greatly improving the efficiency of drug screening and drug analysis. The analysis results are cross validated with DSC technology. For many biotechnology drugs, using the DSF method for rapid mass screening and using DSC technology to orthogonally verify the early screening results of DSF is an effective approach for drug screening and drug development.
DSF has a wide range of applications in protein stability analysis, including but not limited to the following aspects:
Protein thermal stability testing: Most proteins have strict temperature requirements, and the evaluation of protein thermal stability is helpful for the normal expression of protein function. DSF can not only high-throughput determine the thermal stability parameters of proteins, but also evaluate mutations pH、 The influence of factors such as ionic strength on thermal stability, and the selection of factors that can improve thermal stability.
High throughput screening of protein stabilizers and inhibitors: By high-throughput screening of compound ligand libraries, candidate compounds that bind to protein samples are obtained, and the impact of compounds on protein stability is evaluated to screen for protein stabilizers, inhibitors, cofactors, and molecular chaperones.
Research on the mechanism of protein action: such as protein-protein interaction studies, affinity kinetics analysis of newly discovered protein drugs, and thermal stability parameter testing of protein ligand, cofactor, and molecular partner binding.
Research on the inhibition mechanism of small molecule compounds: DSF can be used for high-throughput, matrix based detection of various compounds (compounds bind to some known inhibitors, substrates, products, or cofactors), for screening small molecule compound inhibitors and studying their pharmacological mechanisms.
Protein structure research: Protein crystallization is a very important means of studying protein structure, and using DSF can high-throughput screen the crystallization conditions of proteins.
DSF technology principle:
Differential Scanning Fluorescence (DSF) is an economical, efficient, and easy-to-use biophysical technique that determines the denaturation transition temperature (thermal transition temperature Tm value or chemical denaturation Cm value) of proteins by detecting the corresponding changes in fluorescence emission spectra when the temperature increases or when a denaturing agent is present.
Research has found that aromatic cyclic amino acids in protein molecules experience a shift in the maximum emission spectrum of their excited intrinsic fluorescence when they are in different polar microenvironments, such as hydrophobic or hydrophilic environments. The endogenous fluorescence in proteins mainly comes from aromatic ring amino acids such as tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr), among which tryptophan has stronger endogenous fluorescence.
Taking tryptophan as an example, in the hydrophobic core microenvironment of proteins, its maximum intrinsic fluorescence emission wavelength is around 330nm, while in the hydrophilic polar microenvironment, the maximum intrinsic fluorescence emission wavelength of tryptophan appears around 350nm. Protein thermal or chemical denaturation often leads to a change in polarity of the microenvironment surrounding tryptophan residues, gradually exposing tryptophan, which is usually embedded in the hydrophobic core of proteins, to a hydrophilic environment, resulting in a red shift of the maximum emission wavelength of endogenous fluorescence towards a larger wavelength range.
This method of monitoring endogenous fluorescence changes in tryptophan does not require fluorescent probes or labels, avoiding false positive results such as background fluorescence and non-specific adsorption in traditional exogenous fluorescence DSFs.
When proteins undergo unfolding due to thermal denaturation or chemical denaturation agents (such as guanidine hydrochloride, urea, etc.), measuring the changes in endogenous fluorescence with temperature or denaturation agents can be used to study the unfolding process of proteins. These changes in data can be used to generate melting curves and obtain apparent thermal transition temperature Tm values Tonset、 Van der Waals enthalpy change (Δ H), Gibbs free energy (Δ G), chemical denaturation Cm value, etc. are used for evaluating and predicting protein stability.
Traditional DSF often uses the 350/330 ratio method for data analysis, while SUPR-DSF provides multiple analysis methods. In addition to the ratio method, SUPR-DSF also provides BCM (Barycentric mean), which can achieve better signal-to-noise ratio than the 350/330 ratio method and is more conducive to the analysis of low concentration protein samples.
The main features of the high-throughput protein stability analysis DSF system are:
Using standard 384 microporous plates, no special consumables or capillaries are required
High throughput screening can be completed within one heating process (60-80 minutes)
The testing of a single 384 microporous plate utilizes endogenous fluorescence, without the need for dyes or labels, and is compatible with commonly used biological systems such as UV LED excitation and full spectrum detection
Only 10-30 μ l of sample is needed, with a sample concentration of 0.05-250mg/mL
Obtain key parameters: Tonset, Tm, Δ 919, Δ G, Cm, affinity, etc
Provide rigorous data quality and repeatability
Application of SUPR-DSF system:
The SUPR-DSF system is widely used in various fields such as basic research in life sciences and drug development
Accelerated screening of mutants
Screening and optimization of formulas and pre formulas
Screening of protein crystallization conditions
Batch consistency assessment
Biological similarity assessment
Accelerated stress and forced degradation research
Combined analysis of induced conformational changes
Post translation modification evaluation
Temperature and chemical stability analysis
Technical specifications of high-throughput protein stability analysis DSF system:
Typical application cases--Accelerated screening of mutants
16 samples were compared and screened using SUPR-DSF, including 1 wild-type protein (WT) and 15 single site mutants. Within 1.5 hours, the DSF instrument generated high-quality melting curves for 48 samples (3 replicates per group) (up to 384 wells of data can be generated in the same time). Fitting the data with a model can obtain the melting temperature Tm value and sort and screen the stability.
As shown in Figure 1, compared with WT (blue), the melting curves of three mutants (9, 13, and 14) shifted to the left, indicating a decrease in protein stability; The stability of the remaining 12 mutants has been improved. Among them, mutants 1, 8, and 12 showed the greatest improvement in stability.
Figure 2 shows the melting curves of several representative samples, and it can be observed that some proteins undergo a single thermal transition process (WT protein and mutants 7 and 8), while others (mutants 1 and 14) clearly show two stages of thermal transition, with two inflection points on the melting curve. The data provided by SUPR-DSF can help researchers screen out promising candidates for further research.
Accurately analyze the Fab region of monoclonal antibodies
Obtain differential scanning fluorescence data of NIST mAb (NIST monoclonal antibody standard) using SUPR-DSF, and compare the results with the data obtained by differential scanning calorimetry (DSC). SUPR-DSF can resolve all three domains of NIST mAb: CH2 (69 ° C), CH3 (83 ° C), and Fab region (94 ° C). The highest scanning temperature of SUPR-DSF is 105 ° C, which can accurately measure the melting temperature of abnormally stable Fab regions, while the highest scanning temperature of other DSF platforms is generally only 95 ° C, which can easily lose important information about the unfolding and denaturation of monoclonal antibody Fab regions (Figure 5 (a)).
Compare the normalized data of SUPR-DSF with the DSC results. As shown in Figure 5 (b), the three peaks match each other, demonstrating good consistency between the two techniques. Using SUPR-DSF can easily obtain high-quality protein stability information.
Typical application case - screening of excipients and formulations for accelerated trastuzumab
The formulation of biological therapeutic drugs such as antibodies is the basis for ensuring efficacy, production feasibility, and safety, and also determines the conditions for storage and transportation. Pharmaceutical companies urgently need high-throughput methods to quickly and reliably screen a large number of conditional combinations to determine the optimal formula.
Using SUPR-DSF, researchers screened and analyzed the stability of therapeutic antibody trastuzumab under 96 different conditions, and completed the test within 1.5 hours. The test results are highly consistent with those obtained by differential scanning calorimetry (DSC). If laboratory automation equipment is integrated, thousands of samples can be screened every day.
The DSF melting curve shows two independent transition regions. By fitting the data with the least squares method, the Tonset value, Tm value, and van der Waals enthalpy change (△ H) can be determined. Figures 3 (a, b) show the melting curve and fitting graph of the auxiliary material that destroys/enhances stability, respectively. Figure 4 (a) lists all excipients that can improve antibody stability (compared to the control sample).
SUPR-DSF correctly identified the stability effects of the excipients histidine and trehalose used in marketed trastuzumab, as shown in Figure 4 (b). SUPRDSF data has excellent reliability and consistency, while providing astonishing high-throughput.
Obtaining binding parameters using high-throughput differential scanning fluorescence method
Combining analytical research is a key field in drug development, and the characteristics of interactions and KD values (dissociation equilibrium constants, characterizing the affinity of intermolecular binding) are crucial for the selection of candidate compounds. Researchers need to use multiple complementary principles to screen and determine thousands to tens of thousands of small molecule compounds in the library.
Through SUPR-DSF for molecular interaction analysis, it is not affected by surface effects, mass transport, or buffer refractive index issues, and can provide high-throughput, label free measurements within the range of analyte binding concentrations.
By detecting the stability changes of protein ligand complexes, it can be used to confirm the specificity of binding; The KD value can be calculated by the functional relationship between the changes in fluorescence emission spectra during thermal denaturation and ligand concentration. Even for very weak binding molecules, the stability changes induced by binding can be detected by SUPR-DSF.
By using standard 384 well plates, it is possible to screen the stability of thousands of samples within a day, thereby confirming the binding status.
The binding of ligand (TFMSA) to human carbonic anhydrase I was studied using SUPR-DSF. The melting curve of carbonic anhydrase with TFMSA concentration is shown in Figure 6.
Figure 7 shows the relationship between the Tm value of human carbonic anhydrase I and the concentration of TFMSA. Two KD values were obtained from the fitting, which were 2.1 μ M and 174.2 μ M, respectively
The consistent values in the literature demonstrate that SUPR-DSF can be used as a pre screening or confirmation tool for ligand binding studies and has sensitivity.
Intuitive and concise software
SUPR-DSF software provides intuitive, concise, intelligent, and efficient experimental design and data analysis functions, allowing beginners to quickly get started and providing multiple advanced options for experienced and experienced users.
About ProteinStable
