Abstract
The chemical unfolding (denaturation) assay can be used to calculate the change in the Gibbs free energy of unfolding, ΔG, and inflection point of unfolding, to collectively inform on molecule stability. Here, we evaluated methods for calculating the ΔG across 23 monoclonal antibody sequence variants. These methods are based on how the measured output (intrinsic fluorescence intensity) is treated, including utilizing (a) a single wavelength, (b) a ratio of two wavelengths, (c) a ratio of a single wavelength to an area, and (d) a scatter correction plus a ratio of a single wavelength to an area. When applied to the variants, the three ratio methods showed comparable results, with a similar pooled standard deviation for the ΔG calculation, while the single-wavelength method is shown as inadequate for the data in this study. However, when light scattering is introduced to simulated data, only the scatter-correction area normalization method proves robust. Using this method, common plate-based spectrophotometers found in many laboratories can be used for high-throughput screening of mAb variants and formulation stability studies.
Highlights
Chemical unfolding utilizes chemical denaturants such as guanidine hydrochloride or urea to fully denature a protein or antibody
By using a high-throughput version of this assay, we could potentially differentiate among hundreds to thousands of variants, identifying those variants that are more stable and those variants that are less stable than the parental antibody in a cost, material, and time efficient manner
There are multiple methods reported for using the intrinsic fluorescence intensity output to create chemical unfolding curves and to calculate ΔG
Summary
Chemical unfolding utilizes chemical denaturants such as guanidine hydrochloride or urea to fully denature a protein or antibody. By increasing the concentration of the denaturant, the measured output follows a sigmoidal relationship from a fully folded to a fully unfolded state, typically following a two-state model. From these unfolding curves, an assessment of the protein stability can be obtained by calculating the change in the Gibbs free energy of unfolding, ΔG, and the inflection point, C1/2, of the unfolding curve. Using the ΔG and C1/2 as a grading metric, we are screening antibody variants that have various sequence mutations with the intention of selecting a more stable molecule. By using a high-throughput version of this assay, we could potentially differentiate among hundreds to thousands of variants, identifying those variants that are more stable and those variants that are less stable than the parental antibody in a cost, material, and time efficient manner
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