Binding of the geldanamycin derivative 17-DMAG to Hsp90
For proper folding, many proteins involved in signal-transduction pathways, cell-cycle regulation and apoptosis depend upon the ATP-dependent molecular chaperone Hsp90. Consequently Hsp90 turned out to be an attractive target for cancer therapeutics. In this study we demonstrate the binding of the geldanamycin derivative 17-DMAG to Hsp90 using MicroScale Thermophoresis (MST). The study also highlights the high content information of the MST measurements as one important benefit of MicroScale Thermophoresis.
Interactions of Liposome embeded SNARE Proteins
Competitive Assay Approch: Binding of Small Molecules to the Active Form of p38
Determination of thermodynamic parameters ΔG and ΔH of a small molecule binding to p38 using MST
Characterization of DNA/RNA triplex formation
The concept of double stranded DNA forming triple helical complexes with a third strand of nucleic acids via so called Hoogsteen-hydrogen bonds has been well established for the last 50 years. However, the topic reentered the big stage with recent publications pointing to an existence of these structures in vivo, highlighting their putative role in chromatin organization and transcriptional regulation in a non-coding RNA mediated context.
Binding of Calcium Ions to Synaptotagmin
Binding of Histone peptides to Chromatin assembly factor I (CAF-I) p48 subunit
Using MST to analyse the binding of Nanobodies and Nanobody-Fc fusion proteins to human CD38
Using MST to analyse the binding of the β-Lactamase TEM1 to BLIP
MicroScale Thermophoresis Measurements on in vitro Synthesized Proteins
On a razor’s edge: watching DNaseI cutting DNA into pieces
The Decondensation factor 31 specifically interacts with histones H3 and H4 but not H2A and H2B
The Decondensation factor 31 specifically binds to ssRNA but not to ssDNA or dsDNA
The Decondensation factor 31 binds to mono-nucleosomes
Thermodynamic characterization of DNA hybridization
Analyzing the binding affinity of aptamer quantum dot conjugates to VEGF
Studying the interaction of membrane enzyme PgIB with substrate and inhibitory peptide
Anaerobic MicroScale Thermophoresis reveals the Redox dependency of ferredoxin in mitochondrial Fe/S biogenesis
Studying the interaction of the antibody-drug conjugate SYD985 with an anti-toxin antibody
Interaction of maltose binding protein (MBP) with maltose
One-step, purification-free and site-specific labeling of polyhistidine-tagged proteins for MST
Determination of low-picomolar affinities of sgRNAs and crRNA/tracrRNAs for Cas9
Site-Specific Labeling of Antibodies for MicroScale Thermophoresis
Purification-free Labeling in Whole Cell Lysate and Binding Characterization by MST
Analyzing Thermal Unfolding of Proteins
Detergent Screen for Solubilized membrane proteins-Case study on the SLAC-protein HiTehhA from Haemophilus influenzae
The biophysical characterization of integral membrane protein stability is often challenging due to several factors: First, the expression and purification of membrane proteins is often impeded by low expression levels and protein stability. As a result, yields are usually low and do not allow for a thorough analysis or a screening approach to determine optimal conditions. Second, the use of detergents – which are necessary to solubilize membrane proteins – often introduces artifacts and other secondary effects, and most importantly precludes the use of reporter dyes to monitor protein unfolding. Label free methods – such as DSC or CD spectroscopy – on the other hand require large quantities of proteins, and are limited in throughput.
Thermal Stability Buffer Screening of Therapeutic Antibodies
The development of therapeutic antibodies requires optimal formulations for long-term antibody stability. For this, buffer screening approaches are routinely used, in which the thermal stability of a given antibody in different buffers are tested. The buffers typically vary regarding buffer substances, pH values, salt concentrations and other excipients.
The prerequisite for such screening approaches are high measurement precision, low sample consumption, and high throughput. Moreover, it is highly desirable to measure under native conditions, without dilution of the antibody or the requirement to use reporter dyes or other modifications.
Rapid Quantification of Unfolded Proteins for Quality Control and Optimization of Storage Condistions
The rigorous control of the quality of biological samples is of major importance in pharmaceutical research. On the one hand, drug discovery projects with purified drug targets such as kinases, receptors or integral membrane proteins require a consistent quality of the proteins to ensure successful screening campaigns. On the other hand, a consistent quality of biologicals such as antibodies or other therapeutic proteins is essential in later stages of the drug development process, especially for tests in clinical trials, and is finally required for approval of new drugs by federal agencies.
Analysis of formulation-dependent colloidal and conformational stablity of monoclonal antibodies
The growing number of biological drugs such as monoclonal antibodies (mAbs), as well as the wealth of heterogeneity between mAb variants requires a thorough development process to maximize mAbs compliance with regulation. Therefore, biophysical analytical methods are required already at early stages of the development process to guide and streamline further antibody processing and to predict antibody developability.
Comparison of nanoDSF and µDSC for thermal stability assessment during biopharmaceutical formulation development
The assessment of thermal stability parameters of biologics is an integral part of formulation development in biopharmaceutical research. The ever growing number of biologics in the development pipelines worldwide demands rapid and precise methods to quickly screen large sets of conditions in an easy and straight-forward manner.
In our study, we compare two methods for the detection of thermal unfolding transition temperatures (Tm) of a therapeutic monoclonal antibody (mAb): nanoDSF, which analyzes changes in the fluorescence emission properties of proteins, and differential scanning calorimetry (µDSC), which detects changes in the heat capacity of a protein solution upon unfolding. nanoDSF and µDSC both provide precise and consistent data. nanoDSF in addition overcomes several limitations by µDSC, such as low throughput and high sample consumption, and thus represents the ideal technology for rapid and precise thermal stability screening in biopharmaceutical development.
nanoDSF Thermal Unfolding Analysis of Proteins Without Tryptophan Residues
nanoDSF: Label-free Thermal Unfolding Assay of G Protein-Coupled Receptors for Compound Screening and Buffer Composition Optimization
A thermal unfolding based assay using low volume differential intrinsic tryptophan scanning fluorimetry (nanoDSF) was applied to study the stabilizing effects of ligands on G-protein-coupled receptors (GPCRs). GPCRs are the fourth largest superfamily in the human genome and are the largest class of targets for drug discovery. The system has been validated using human adenosine A2A receptor (A2AR). A2AR binds natural (adenosine and caffeine) and synthetic ligands with different affinities to mediate a variety of physiological and pharmacological responses. Several well characterized ligands were used for the unfolding experiments. The ΔTm shift values obtained from nanoDSF analysis and traditional ligand binding studies correlate well with each other. We further characterized a second human GPCR target (test-GPCR) for which traditional cysteine-reactive DSF has been problematic. nanoDSF demonstrated that small molecule ligands can stabilize the detergent-solubilized receptor, thus showing the target GPCR is active in a selected detergent and lipid-free environment. In addition, we report a buffer composition screen to further stabilize the receptor in its detergent environment for biophysical assays.
Chemical and Thermal Stability Screening of an IgG1-Antibody
The main purpose in formulation development of biologicals, such as monoclonal antibodies, is to establish optimal conditions for long-term stability of the protein.
Here we used the Prometheus NT.48 instrument by NanoTemper Technologies to perform a buffer screening for an IgG1-Antibody. This instrument uses nanoDSF technology to measure thermal and chemical stability as indicators to predict the best buffer conditions for the protein.
The results show that thermal and chemical unfolding, together with short-term stability studies, provides a complementary screening tool for finding optimal buffer conditions.
Rapid and Precise Biosimilar Candidate Profiling by nanoDSF
The development of biosimilars requires extensive physicochemical characterization of biosimilar candidate molecules which should match the quality profile of the reference molecule (originator).
Here we use a novel method of thermal unfolding profiling to rapidly screen a variety of Fc-fusion protein biosimilar candidates. The best-in-class precision of the NanoTemper Technologies Prometheus NT.48 nanoDSF instrument allowed for the ranking of biosimilar candidates according to the comparability of their unfolding profiles to the reference molecule. The results were in excellent agreement with conventional screening methods, while dramatically reducing sample consumption and measurement times. Thus, nanoDSF (miniaturized differential scanning fluorimetry) is a new and powerful tool for rapid screening approaches in biosimilar development. It enables narrowing down the number of promising candidates in hours instead of days or weeks.
Getting the Full Picture: Predicting the Aggregation Propensity of mAbs Using Chemical and Thermal Denaturation on a Single, Fully Automated Platform
One of the most important parameters in the development of therapeutic biologics is their long-term stability. While after purification being seemingly stable in a variety of formulations, many antibodies display very slow aggregation kinetics over time. This gradual aggregation could thus far only be evaluated by monitoring monomer contents and aggregates over months or even years. Predictive methods are therefore urgently needed to speed up the development of biologics.
Here we demonstrate that the Prometheus NT.Plex by NanoTemper Technologies can be used for predicting long-term stability in only a few hours. The approach uses a combination of thermal and chemical unfolding analysis in a high-throughput setting. We show that chemical denaturation is a tool which can determine folding enthalpies of monoclonal antibodies (mAbs) to predict their long-term stability in formulation screenings. Using the Prometheus NT.Plex nanoDSF instrument with aggregation detection optics, we screened 5 formulations at different mAb concentrations for their thermal and chemical stability. The obtained unfolding data correlates with long-term turbidity and monomer content over time, showing that the Prometheus NT.Plex can be used to rapidly predict the long-term stability of biologics within 1 day.
nanoDSF Thermal Unfolding Analysis of a Membrane-bound Esterase in Various Detergents
Use of iFormulate™ and nanoDSF for Fast and Precise Protein Formulation Development
One of the challenges in protein formulation is the simultaneous evaluation of multiple key formulation variables in a rational fashion. The Design of Experiments (DOE) approach has gained significant popularity in protein formulation as well as other process development activities.
HTD Biosystems has developed a robust DOE approach for protein formulations referred to as iFormulate™ that utilizes advanced response surface quadratic modeling to evaluate the effects of four formulation variables in a multivariate fashion. The procedure allows for rapid identification of stable protein formulations, using only small amounts of material.
By analyzing the thermal stability of a model protein, Lysozyme, using the Prometheus NT.48 in conjunction with the iFormulate™ DOE approach, we demonstrate that ideal formulations can be identified in as little as 35 minutes with minimal sample consumption.
The DOE analysis of the precise and reproducible data generated by NanoTemper Technologies Prometheus NT.48 from 25 trial formulations resulted in the identification of the formulation design space for lysozyme formulations with high melting temperatures (Tms). The formulations can be rationalized using Quality by Design principles from the DOE analysis. Thus, by using the predictions from the DOE output and high-precision thermal stability analysis by nanoDSF, stable formulations of proteins can be generated and validated with unprecedented speed and precision.