Fluorimetry Pdf Notes
Shu Manwill
Measuring the thermal stability of three AAV serotypes, AAV2, AAV5, and AAV6, using the easy plate reader technology for protein stability screen: the SUPR-DSF. The SUPR-DSF measured the intrinsic fluorescence of each serotype during a thermal ramp to generate melt curves. In addition, we performed a concentration dilution series on AAV2 and AAV6 to determine the minimal sample requirements. All samples were performed at a well volume of 10μL in triplicate on a single 384-well microplate, emphasising the high-throughput possibilities of the instrument.
Analysing the statistical distribution of over 6000 repeat lysozyme samples gave accurate Tm values while providing a low standard deviation of only 0.14C. The SUPR-DSF, therefore, has great utility within protein stability screening, connecting to supporting technologies, minimising handling errors and risk, and offering more samples with lower sample usage.
The NISTmAb is a widely characterised monoclonal antibody intended to be used as a reference molecule in the development of novel technology for therapeutic protein characterisation. In this application note, we use the SUPR-DSF to acquire differential scanning fluorimetry (DSF) data on the NISTmAb and compare the results against differential scanning calorimetry (DSC). The SUPR-DSF resolved all three domains of the NISTmAb: CH2 (69C), CH3 (83C), and the unusually high melting temperature of the Fab domain (94C). In addition, the apparent melting temperatures agreed exceptionally well with the literature results. These results validate you can easily obtain high-quality protein stability information with the SUPR-DSF.
In this study, we show the analysis of a thermal denaturation-based formulation screen of the commercially available therapeutic antibody Trastuzumab in 96 different conditions with the
SUPR-DSF instrument. Along with screening the stabilising agents, confidence in the results is gained as there is consistency with both Differential Scanning Calorimetry and the formulation used for the commercial drug Herceptin. This screen was directly measured in a single 384-well microplate in less than 1.5 hours. This high throughput can be leveraged further through lab automation integration to screen thousands of samples per day.
To illustrate the use of the SUPR-DSF for variant comparison and selection in early-stage discovery, we have used a model protein system to compare 16 analogues and identify the most stable ones for further processing.
The use of the SUPR-DSF to study binding interactions has demonstrated that this technique offers a viable and unique solution. The SUPR-DSF offers an intrinsic fluorescence-based high-throughput methodology that is free in solution, with all measurements carried out directly in the 384-well plate-based format. The SUPR-DSF rapidly provided highly precise data with excellent sensitivity in the study of Carbonic Anhydrase with TFMSA. The values obtained agree with previously published literature values for KD and prove that the SUPR-DSF can be used as either a pre-screening or conformational tool for ligand binding studies.
In this application note, the SUPR-CM fluorescence plate reader was used to determine the aggregation propensity of the model protein lysozyme in two different buffer solutions in order to illustrate how ICD can be used to assess which buffer condition would result in fewer aggregates.
Forced degradation studies are routinely employed during the development of new biologics. Along with providing evidence for the mechanisms by which a mAb can degrade, the change in stability mimics the observations when comparing different antibody constructs.
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.
Abstract. The atmosphere simulation chamber SAPHIR at the Research Centre Jlich was used to test the suitability of state-of-the-art analytical instruments for the measurement of gas-phase formaldehyde (HCHO) in air. Five analyzers based on four different sensing principles were deployed: a differential optical absorption spectrometer (DOAS), cartridges for 2,4-dinitrophenylhydrazine (DNPH) derivatization followed by off-line high pressure liquid chromatography (HPLC) analysis, two different types of commercially available wet chemical sensors based on Hantzsch fluorimetry, and a proton-transfer-reaction mass spectrometer (PTR-MS). A new optimized mode of operation was used for the PTR-MS instrument which significantly enhanced its performance for online HCHO detection at low absolute humidities.
The instruments were challenged with typical ambient levels of HCHO ranging from zero to several ppb. Synthetic air of high purity and particulate-filtered ambient air were used as sample matrices in the atmosphere simulation chamber onto which HCHO was spiked under varying levels of humidity and ozone. Measurements were compared to mixing ratios calculated from the chamber volume and the known amount of HCHO injected into the chamber; measurements were also compared between the different instruments. The formal and blind intercomparison exercise was conducted under the control of an independent referee. A number of analytical problems associated with the experimental set-up and with individual instruments were identified, the overall agreement between the methods was fair.
This document provides an introduction to fluorimetry and phosphorimetry. It defines fluorescence as the emission of visible light when certain substances are exposed to light, while phosphorescence is the continued emission of light even after the light source is removed. The principle involves excitation of electrons from the highest occupied to the lowest unoccupied molecular orbital. Factors that affect fluorescence and phosphorescence include concentration, oxygen levels, pH, temperature, substituents, scatter, and adsorption. Applications include determination of various compounds in samples like urine, serum, food, and more.Read less
High throughput protein stability screening of construct libraries is an established technique used to quickly identify the best candidates for downstream processing. Having the most stable form of the protein, for example, an engineered antibody is a solid basis for further optimisation work before forwarding the candidates to the next steps of development. Often involving thousands of early candidates this can be a time-consuming and expensive process. With the SUPR range, of thermal and chemical melt options, stability screening has never been more efficient. High throughput differential scanning fluorimetry using microplate technology, in either 96- or 384-well formats with the inherent ability to prepare the samples in automated systems already being used for other steps of the process means a far higher number of constructs can be screened quickly and without the added workload and cost associated with other techniques. Particularly important at this stage is the low sample usage as there are always limited amounts of samples. Use of thermal melts with the SUPR-DSF yields the best options in a matter of a few hours, not days and further, preferred candidates can be screened orthogonally with the data richness of SUPR-CM chemical melts.
Once candidates have passed through early development and optimisation stages, it is important to understand how they will be stored, transported, and what safe lifetimes they can have before use. Testing multiple formulations under a range of conditions is essential to the understanding and prediction of long-term stability. Having automatable microplate reader technology combining thermal and chemical melt options can quickly identify the best conditions applied by ranking all formulations in stability thus suggesting which will be most stable in storage. Fast, data-rich information gives insight into both thermodynamic and kinetic stabilities, leading to confidence in decisions about which formulations should be used.
Binding analysis studies are a crucial area of drug development when either screening for hits or working on an optimisation study. The characterisation of the interaction between protein target and ligand, and the KD (Dissociation constant) parameter are critical in selecting the best candidates for further development. With high data quality, false positives or negatives can be eliminated and thus avoid issues of progressing candidates with non-specific binding. Orthogonal approaches are frequently employed to ensure the robustness of decision-making based on the best data available.
Interactions analysis by the SUPR-DSF offers label-free measurements across a wide analyte binding concentration range. The measurements are free in solution and are not influenced by surface effects, mass transport or refractive index buffer issues thus providing the perfect complementary data to more traditional binding analysis techniques. With high throughputs, low sample usage, and easy processing in 384-well plate format, orthogonal data has never been easier.
To implement the Qubit Flex SAE firmware Solution to support the technical 21 CFR Part 11 compliance portion on the Qubit Flex Fluorometers, the following components need to be installed, activated, and communicating with each other.
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