Protein Stability Screening via Differential Scanning Fluorimetry
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Solution Overview
Problem
Current methods for assessing the physiochemical properties of proteins, particularly therapeutic proteins, require significant quantities and are not suitable for high throughput screening, especially under low pH conditions relevant to viral inactivation processes, leading to challenges in predicting stability and manufacturability.
Innovation Solution
The use of differential scanning fluorimetry (DSF) with hydrophobic fluorescent dyes to assess protein stability by heating samples at varying temperatures and pH levels, allowing for the detection of temperature transitions and calculation of weighted shoulder and leader scores to predict protein stability, even with limited protein quantities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional techniques such as differential scanning calorimetry (DSC) are used to interrogate physiochemical properties of proteins, then accurate measurement of protein stability is achieved, but large quantities of protein (gram quantities) are required
Solution Approach 1:
The patent replaces the mechanical/thermal measurement system of DSC with an optical detection system using differential scanning fluorimetry (DSF). The DSF system uses fluorescent dyes that bind to hydrophobic regions of proteins, and fluorescence intensity changes are detected optically as proteins unfold with temperature, enabling high-throughput screening with minimal protein quantities while maintaining stability assessment capability
Solution Approach 2:
The patent changes the detection parameter from direct thermal measurement (DSC) to fluorescence intensity measurement (DSF). By using temperature-sensitive fluorescent dyes that change emission properties upon protein unfolding, the system achieves equivalent stability information with much smaller sample volumes, enabling screening of multiple protein variants simultaneously
2Productivity
If high throughput screening is implemented to assess protein stability under low pH conditions, then productivity is improved, but measurement precision may be compromised due to limited protein quantities
Solution Approach 1:
The patent implements high-throughput screening by replacing traditional low-throughput methods with DSF using 96-well or 384-well plates. The optical detection system can simultaneously monitor fluorescence changes in hundreds of samples, each containing minimal protein (50-100 μg), enabling comprehensive stability assessment of multiple protein variants under various low pH conditions without sacrificing measurement quality
Solution Approach 2:
The DSF system serves multiple functions simultaneously: it measures thermal stability, pH stability, and aggregation propensity using the same fluorescent dye-based approach. This universal methodology allows the system to screen for multiple stability parameters across numerous protein variants in parallel, maintaining precision while dramatically increasing productivity
3Device complexity
If ambiguous peak-fitting approaches are used to analyze DSF data, then device complexity is reduced, but measurement precision deteriorates due to ambiguous results
Solution Approach 1:
The patent extracts the critical stability information from complex DSF thermograms by identifying and isolating the melting temperature (Tm) as the primary parameter. Rather than attempting to fit entire complex curves with multiple parameters, the method extracts the key Tm value where the fluorescence transition occurs, providing clear, unambiguous stability metrics that are directly comparable across samples
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables high throughput assessment of protein stability at low pH conditions, predicting suitability for viral inactivation and manufacturability with minimal protein quantities, avoiding ambiguous peak-fitting approaches and providing accurate metrics for thermal stability.
Implementation Method 1
heating to a continuous set of temperatures in the range of about 20° C. to about 95° C. a first reaction mixture comprising a hydrophobic fluorescent dye and the protein of interest
Implementation Method 2
DSF uses an extrinsic hydrophobic dye to monitor protein thermal unfolding
Implementation Method 3
assess protein unfolding temperatures as a protein is subjected to an increasing thermal gradient
Data Source
AI summary
Disclosed are methods for interrogating the physiochemical properties of a protein of interest, including method for predicting the stability of a protein at low pH, such as may be encountered during a manufacturing viral inactivation step.