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应用案例

MO-001: Protein-Small Molecule Interaction analysis

Binding of the geldanamycin derivative 17-DMAG to Hsp90

AppNote NT-001

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.

MO-002: Protein-Vesicle Interaction Analysis

Interactions of Liposome embeded SNARE Proteins

AppNote NT-002
SNARE protein complexes play a key role in membrane fusion processes, e.g. the fusion of synaptic vesicles with the pre-synaptic plasma membrane in response to elevated Ca2+ levels. This study aimed to demonstrate the use of MicroScale Thermophoresis for the detection of protein-protein interaction in more complex assay systems like in this case the interaction of membrane proteins embedded into liposomes.

MO-003: p38 competitive binding assay

Competitive Assay Approch: Binding of Small Molecules to the Active Form of p38

AppNote NT-003
p38α is considered as the key isoform involved in modulating inflammatory response in rheumatoid arthritis and inflammatory pain and is therefore a key target for compound screening in pharmaceutical industry. In this study we demonstrate the use of MicroScale Thermophoresis to perform an essentially label-free measurement of small molecule binding to protein targets by using a competitive approach. This approach therefore allows screening projects aiming to identify and characterize site-specific binders or binders allosterically influencing a specific binding site out of a compound library. Furthermore this approach also can be used to screen for compounds that interrupt protein-protein, protein-peptide or protein-nucleic acid interactions.

MO-004: Protein-Small Molecule Interaction analysis

Determination of thermodynamic parameters ΔG and ΔH of a small molecule binding to p38 using MST

AppNote NT-004

MO-005: Nucleic Acid Interaction Analysis

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.


In this study we characterize the formation of such complexes in vitro, using MicroScale Thermophoresis (MST). The study also highlights the high content information of the MST measurements as one important benefit of MicroScale Thermophoresis.

MO-006: Protein-Ion Interaction Analysis

Binding of Calcium Ions to Synaptotagmin

AppNote NT-006
The synaptic vesicle protein synaptotagmin 1 is the main calcium sensor of neuronal exocytosis. Calcium binds to its cytosolic portion that consists of tandem C2-type domains. In this work we show that MicroScale
Thermophoresis is a valuable tool to measure binding of ions to proteins, with and without the use of a fluorescent label.

MO-008: Protein - Peptide Interaction Analysis

Binding of Histone peptides to Chromatin assembly factor I (CAF-I) p48 subunit

AppNote NT-007
p48, the small subunit of chromatin assembly factor 1 (CAF-1), is a member of a highly conserved subfamily of WD-repeat proteins. There are at least two members of this subfamily in human (p46 and p48). p48 copurifies with a chromatin assembly complex (CAC), which contains the three subunits of CAF-1 (p150, p60, p48) and the Histones H3 and H4, and promotes DNA replication-dependent chromatin assembly.
In this study we analyze the binding of H3 and H4 peptides to p48 using MicroScale Thermophoresis (MST). The study also highlights the high content information of the MST measurements as one important benefit of MicroScale Thermophoresis.

MO-011: Antibody-Antigen Interaction Analysis

Using MST to analyse the binding of Nanobodies and Nanobody-Fc fusion proteins to human CD38

AppNote NT-011
Antibody affinities play an important role in diagnostic, basic research and medical applications. Conventional antibodies are composed of two heavy and two light chains. Both chains contribute to binding of the antigen by their variable domains. In addition to conventional antibodies, llamas produce functional heavy chain antibodies that lack light chains and interact with antigens solely via the variable domain of the heavy chain, designated VHH. Recombinant VHHs are also called single domain antibodies or Nanobodies because of their size in the nm range. In this study we compared the binding of Nanobodies and Nanobody-Fc fusion proteins to human CD38 using MicroScale Thermophoresis (MST). This study highlights the potential of MST Technology in affinity determination of antibody-antigen interactions.

MO-012: Protein-Protein Interaction Analysis in Different Buffer Systems

Using MST to analyse the binding of the β-Lactamase TEM1 to BLIP

AppNote NT-012
The analysis of protein-protein interactions plays an important role in understanding many biological processes. MicroScale Thermophoresis (MST) permits a robust and fast analysis of protein-protein interactions even in highly complex biological liquids. Here we have investigated the binding of TEM1 β – Lactamase to BLIP (β – Lactamase Inhibitory Protein) using MicroScale Thermophoresis. The experiments were performed as in 50 % mammalian cell lysate, as well as in standard buffer. The TEM1-BLIP system is very well characterized by various biophysical methods, such as Surface Plasmon Resonance (SPR).


The role of residue substitutions at the interface of the proteins was easily studied using TEM1 and BLIP mutants. Dissociation constants (Kd) determined for TEM1 and BLIP show an excellent agreement with previously published data.

MO-013: Protein-Protein Interaction Analysis

MicroScale Thermophoresis Measurements on in vitro Synthesized Proteins

AppNote NT-013
Nowadays, especially through the advent of systems biology, and the ability to generate predictive models, it is even more important to transform biology from a qualitative to a quantitative science. Interactions between proteins not only need to be identified, but their equilibrium rate constants need to be determined as well. Here we describe a very elegant and simple system to obtain quantitative data for protein-protein interactions using cell-free protein biosynthesis in combination with MicroScale Thermophoresis. We have been able to characterize the interaction of Calmodulin with Ca2+ as well as its ligand M13 straight in the in vitro synthesis reaction. Furthermore, we are demonstrating the generic applicability of this approach with another set of proteins, an Antibody fragment and Cyclophilin A. All this work has been done without tedious expression and prior purification of the interacting proteins.

MO-014: Enzyme kinetics

On a razor’s edge: watching DNaseI cutting DNA into pieces

AppNote NT-014
MicroScale Thermophoresis (MST) has been established to be a powerful technique to study interactions of molecules in equilibrium state and to determine dissociation constants. In this application note, we present that employing MST we could monitor DNase I endonuclease activity in real time. We chose two different approaches to investigate nuclease activity: in the first approach we directly measured nuclease activity in the same capillary in time steps of 3 s. In the second approach we stopped the reaction chemically after different incubation periods and then performed MST measurements. In both experiments we detect a time dependent change in thermophoresis we can assign to DNase I endonuclease activity. Thus, we could demonstrate that MST is also a valid method to visualize enzyme kinetics.

MO-015: Characterization of a Protein - Histone Interaction

The Decondensation factor 31 specifically interacts with histones H3 and H4 but not H2A and H2B

AppNote NT-015
Histone proteins are associated with DNA and form a nucleoprotein structure termed chromatin. Eukaryotic genomes are packed into chromatin for storage and transport of the DNA. To regulate access to DNA for various essential processes such as transcription and translation, histone modifying, DNA modifying and chromatin binding proteins specifically interact with chromatin structures. Here we characterize the specific interaction of the Decondensation factor 31 (Df31) with core histones. Df31 was recently shown to form a complex with snoRNAs and chromatin, to generate accessible higher order structures of chromatin.

MO-016: Characterization of Protein - Nucleic Acid Interactions

The Decondensation factor 31 specifically binds to ssRNA but not to ssDNA or dsDNA

AppNote NT-016
Histone proteins are associated with DNA and form a nucleoprotein structure termed chromatin. The chromatin structure allows the eukaryotic genome to be stored and transported but also needs to be accessible to various crucial DNA dependent processes Thus, histone modifying, DNA modifying and chromatin binding proteins specifically interact and regulate chromatin.. Interestingly, many of the chromatin modifying proteins also display RNA binding capacity. Here we characterize the specific interaction of the Decondensation factor 31 (Df31) with nucleic acids. Df31 was recently shown to form be part of a RNA dependent regulating mechanism influencing chromatin structure and function.

MO-017: Characterization of a Protein - Nucleosome Interaction

The Decondensation factor 31 binds to mono-nucleosomes

AppNote NT-017
Chromatin, the packaging form of the genome possesses two fundamental functions. On the one hand it compacts the DNA to fit into the nucleus and on the other hand it allows access to the underlying sequences for essential DNA dependent processes. Nucleosomes represent the basic structural component of chromatin. Here we characterize the specific interaction of the Decondensation factor 31 (Df31) with mono-nucleosomes. Df31 was recently shown to form a complex with snoRNAs and chromatin, to generate accessible higher order structures of chromatin.

MO-018: Thermodynamic characterization of DNA hybridization

Thermodynamic characterization of DNA hybridization

AppNote NT-018
Here we report a thermodynamic analysis of biomolecular interactions using MicroScale Thermophoresis (MST). Using DNA hybridizationwith fluorescently labeled oligonucleotides as a well-characterized model system, we monitor the shift in the dissociation (Kd) constant of the interaction over a range of temperatures and calculate the free enthalpy (ΔH) and entropy (ΔS) for the hybridization reaction. The obtained values were in excellent agreement with theoretically calculated values, demonstrating that MST is feasible to perform thermodynamic analysis of biomolecules.

MO-019: Protein-Quantum Dots Interaction Analysis

Analyzing the binding affinity of aptamer quantum dot conjugates to VEGF

AppNote NT-019
Aptamers are single-stranded oligonucleotides which fold into a three-dimensional structure allowing the specific recognition of a molecular target. Therefore, they might be an alternative to antibodies as detection molecules in biosensors. Aptamers are well suited for this application since they can be directed against almost every molecular target, and show high binding affinities to their corresponding binding partner. Aptamers with a terminal amino-group can be coupled to organic or inorganic fluorophores via amide bonds to enable their optical detection. In this study Vascular Endothelial Growth Factor (VEGF)-binding aptamers were conjugated with quantum dot molecules. This immobilization may inhibit the correct aptamer folding and consequently lead to a declined target-binding activity. Therefore, MST technology was used to determine whether quantum dot conjugation affects the VEGF-binding affinity of aptamers.

MO-024: Membrane enzyme PgIB interact with substrate and inhibitory peptide

Studying the interaction of membrane enzyme PgIB with substrate and inhibitory peptide

AppNote NT-024
During the catalytic cycle of enzymatic reactions, the recognition of a specific substrate by the enzyme is one of the most important steps, and the analysis of the affinity between enzyme and substrates and/or inhibitors is an important aspect in mechanistic biochemistry. Here, we have performed an analysis of the binding affinity between two different peptides and the bacterial oligosaccharyltransferase, PglB. Using labeled peptides, we have performed MicroScale Themophoresis experiments to determine the Kd values for the interaction between PglB and these peptides. The results show a high concordance with the values previously reported for the same interaction determined by fluorescence anisotropy, showing that MST is a suitable tool for the study of membrane protein-substrate interactions in detergent solutions.

MO-026: Anaerobic MicroScale Thermophoresis

Anaerobic MicroScale Thermophoresis reveals the Redox dependency of ferredoxin in mitochondrial Fe/S biogenesis

AppNote NT-026
Maturation of iron-sulphur (Fe/S) proteins involves a complex biosynthetic machinery. Here we investigate the interaction of one component, Yah1, with its interaction partners apo-Isu1 and Nfs1-Isd11. Using anaerobic MicroScale Thermophoresis (MST) we show the redox dependency of the interaction between Yah1 and Isu1 as well as between Yah1 and Nfs1-Isd11. Reduced but not oxidized Yah1 tightly interacts with apo-Isu1 indicating a dynamic interaction between Yah1−apo-Isu1.

MO-027: Antibody-Drug Conjugates (ADCs)

Studying the interaction of the antibody-drug conjugate SYD985 with an anti-toxin antibody

AppNote NT-027
Antibody-drug conjugates (ADCs) have been recognized as a promising new class of therapeutic agents for the treatment of cancer (1). In the generation of ADCs, cytotoxic small molecules are covalently attached to therapeutic antibodies thereby increasing their tumor cell killing capability. We have performed MicroScale Thermophoresis experiments to determine the Kd values for the interaction between fluorescently labeled antibody-drug conjugate (SYD985) and an anti-toxin (drug) antibody in phosphate buffer as well in human plasma. The data show that MicroScale Thermophoresis is a powerful tool for the study of antibody-antigen interactions in standard buffers as well as complex bioliquids

MO-028: Label-free Protein-Sugar Interaction Analysis

Interaction of maltose binding protein (MBP) with maltose

AppNote NT-028
Protein-sugar interactions are essential for nutrient transport and metabolism in all types of cells. Here, we investigate the interaction of maltose binding protein (MBP) and its substrate, maltose, drawing upon label-free MicroScale Thermophoresis (MST) as the ideal technique. We also show that sample viscosity did not affect the binding assay, and discuss how MST is not limited by sample viscosity.

MO-029: One-step, purification-free and site-specific labeling of his-tagged proteins

One-step, purification-free and site-specific labeling of polyhistidine-tagged proteins for MST

AppNote NT-029
NanoTemper Technologies uses the popular polyhistidine-tag as target for an unparalleled one step, purification-free labeling of proteins for MST experiments. The labeling strategy employs a high affinity multivalent nitrilotriacetic acid (NTA) derivative conjugated with the MST-optimized NT-647 dye (RED-tris-NTA). As the hexahistidine tag (His6) provides binding sites for three NTA moieties, RED-tris-NTA is perfectly suited for non-covalent, stable, highly selective labeling with a 1:1 stoichiometric ratio. The labeling can be performed with minute amounts of either purified biomolecules or directly in the cell lysate.

MO-030: High-Affinity Protein-RNA Interactions

Determination of low-picomolar affinities of sgRNAs and crRNA/tracrRNAs for Cas9

AppNote NT-030
Recent advances in genome engineering technologies based on the RNA-guided CRISPR endonuclease Cas9 are enabling systematic manipulation of genome function in a variety of organisms, ranging from bacteria and archaea to humans. Cas9 is guided to specific locations within a genome by a short RNA search string. Since genome editing leads to permanent modifications within a genome, the targeting specificity of Cas9 nucleases is of particular importance, especially for clinical application and gene editing.


In this work, we demonstrate the versatility of MicroScale Thermophoresis (MST) to determine the binding affinities of various single-guide RNA (sgRNA), duplex of CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) constructs with Cas9. The MST assay is superior to classical methods like electrophoretic mobility shift assay (EMSA) because it allows effortless Kd determination free in solution. Additionally, MST provides excellent sensitivity while consuming a small amount of non-hazardous fluorescent-labeled oligos or protein. We analyzed several modified RNAs, some of which were labeled with the fluorophore Cy5. Using NanoTemper Technologies Monolith NT.115Pico instrument, we determined the affinities of Cas9 with RNAs differing in length and chemical modification pattern. All interactions were in the lower picomolar range, the highest measured affinity was 1.0 pM.

MO-031: Site-specific labeling of antibodies

Site-Specific Labeling of Antibodies for MicroScale Thermophoresis

Site-specific conjugation of a dye to antibodies is useful for producing highly homogeneous material for use in downstream affinity experiments. This application note describes a labeling procedure that combines the selective reduction of antibody interchain disulfides and their labeling with maleimide-based chemistry. Different antibodies conjugated by this approach yield high-quality MST affinity data for interactions with both antigens as well as Fc receptors.

MO-032: Purification-free labeling in whole cell lysate

Purification-free Labeling in Whole Cell Lysate and Binding Characterization by MST

AppNote NT-032
For researchers performing biophysical analysis of proteins, a common hurdle encountered is having sufficient amounts of materials with the appropriate purity to perform detailed analysis. Here we demonstrate the utility of a RED-tris-NTA dye from NanoTemper Technologies that can be used to specifically label His-tagged proteins for MicroScale Thermophoresis (MST) binding studies directly in cell lysates. The procedure requires minute amounts of sample, can be carried out without additional lab equipment and accurate Kd measurements are obtained within 45 minutes from cell lysis to measurement. As an example, we measured the affinity of a small molecule inhibitor to His-tagged p38α kinase expressed in mammalian cells. Our data demonstrate that MST assays are a rapid and cost-effective method for determination of affinities using unpurified proteins, and thus serves as a powerful tool in the early drug discovery, especially for proteins that are difficult to purify.

PR-001: Analyzing Thermal Unfolding of Proteins

Analyzing Thermal Unfolding of Proteins

A detailed analysis of protein stability is a prerequisite for both, the basic understanding of protein folding mechanisms as well as for the successful development of biologicals in the pharmaceutical industry. Here we demonstrate the performance of the new Prometheus NT.48 instrument, which detects intrinsic protein fluorescence changes upon thermal or chemical unfolding of up to 48 samples in parallel.

PR-002: Detergent Screen for solubilized membrane 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.


Here we use the 10 transmembrane-helix protein HiTehA, a protein of the slow anion channel family, to present label-free, native DSF as the method of choice to perform rapid and precise detergent screening projects for a solubilized membrane protein.

PR-003: Thermal Stability Buffer Screening of Therapeutic Antibodies

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.


Here we present nanoDSF, a label-free, native DSF technique, as the method of choice to perform rapid and precise buffer screening projects for therapeutic antibodies during the drug development process.

PR-004: Rapid Quantification of Unfolded Proteins for Quality Control and Optimization of Storage Conditions

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.


Here we demonstrate how the Prometheus NT.48 can be used to investigate the long-term stability of proteins as well as to determine optimal handling conditions by quantifying the fraction of unfolded proteins within seconds.

PR-005: colloidal and conformational stability of monoclonal antibodies

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.


In this case study, we demonstrate how the Prometheus NT.48 can be used to predict long-term mAb stability in a formulation screen by simultaneous quantification of both, conformational and colloidal stability of biologicals in thermal gradients.

PR-006: Comparison of nanoDSF and μDSC for thermal stability assessment

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.

PR-007: Thermal Unfolding Analysis of Proteins Without Tryptophan Residues

nanoDSF Thermal Unfolding Analysis of Proteins Without Tryptophan Residues

The nanoDSF (minituarized differential scanning fluorimetry) technology is a powerful method to determine the thermal and chemical stability of proteins by following changes in fluorescence, where the amino acid tryptophan serves as the main source for the fluorescence signal.  We analyzed the stability of the light-oxygenvoltage (LOV) protein of the marine phototrophic α-proteobacterium Dinoroseobacter shibae (DsLOV), which does not contain any tryptophan residues. Further, we examined the impact of various mutations on DsLOV protein stability. This study demonstrates the ability of the Prometheus NT.48 to analyze protein stability in samples that do not contain any tryptophan residues.

PR-008: Compound screening and buffer composition optimization of GPCR

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.


Based on our results, we show that the nanoDSF technology will allow the development of an automated screening platform in a label-free environment to evaluate a large number of compounds for lead discovery and to improve receptor stability for biophysical assays by screening buffer conditions.

PR-009: Chemical and Thermal Stability Screening of an IgG1- Antibody

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.

PR-010: Rapid and Precise Biosimilar Candidate Profiling

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.

PR-011: Predicting the Aggregation Propensity of mAbs Using Chemical and Thermal Denaturation

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.


PR-012: Stability of membrane-bound proteins

nanoDSF Thermal Unfolding Analysis of a Membrane-bound Esterase in Various Detergents

nanoDSF, the miniaturized differential scanning fluorimetry technology, is a revolutionary method to determine the thermostability of proteins by following changes in their intrinsic fluorescence. In this comparative study, the Prometheus NT.48 was used to determine the thermal stability of the membrane esterase PA2949 from Pseudomonas aeruginosa in presence of various detergents. The detergent type strongly affected enzyme thermal stability, which moreover correlated with enzyme activity. Thus, the Prometheus NT.48 can be used not only to rapidly screen for optimal purification conditions for enzymes, but also to evaluate enzyme activities based on their conformational stability in presence of detergents.

PR-013: Fast and precise protein formulation development

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.