Methods for detecting and determining protein structures and stability in fluids, including biological fluids
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsMichael Rosconi, Nina Liu, Erica Pyles
Life of this patent
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Abstract
The present inventions provide methods for detecting and determining protein structures and stability, including heteromeric protein complexes, in biological fluids, such as serum and other bodily fluids. Systems for performing the methods also are provided.
Claims30
A method for characterizing one or more complex types comprising at least one protein of interest using asymmetric flow field-flow fractionation (A4F), wherein the method comprises the steps of:
- (A) fractionating a first aliquot comprising complexes in a fractionation buffer using A4F, and determining at least one of molar mass, stoichiometry, and size distribution of the complexes in the first aliquot using Multi-Angle Light Scattering (MALS);
- and
- (B) fractionating a second aliquot comprising the complexes bound with a fluorescence labeled detection reagent in a biological fluid using A4F, and detecting the protein of interest in the complexes using a fluorescence detector,
- wherein (A) and (B) can be performed consecutively in any order or simultaneously;
- and
- (C) comparing the elution time profiles from (A) and (B) to characterize complex types.
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A method for evaluation stability of a protein in a biological fluid using asymmetric flow field-flow fractionation (A4F), wherein the method comprises the steps of:
- (A) fractionating a first aliquot comprising the protein in a fractionation buffer using A4F, and determining size distribution of the protein in the first aliquot using Multi-Angle Light Scattering (MALS);
- (B) fractionating a second aliquot comprising the protein bound with a fluorescence labeled detection reagent in a biological fluid using A4F, and detecting the protein using a fluorescence detector,
- wherein (A) and (B) can be performed consecutively in any order or simultaneously;
- and
- (C) comparing the elution time profiles from (A) and (B).
A method for characterizing one or more complex types comprising at least one protein of interest using fractionation, wherein the method comprises the steps of:
- (A) fractionating a first aliquot comprising the complexes in a fractionation buffer using asymmetric flow field-flow fractionation (A4F) or size exclusion chromatography (SEC), and determining at least one of molar mass, stoichiometry, and size distribution of the complexes in the first aliquot using Multi-Angle Light Scattering (MALS);
- (B) fractionating a second aliquot comprising the complexes bound with a fluorescence labeled detection reagent in a biological fluid using A4F or SEC, and detecting the protein of interest in the complexes using a fluorescence detector,
- wherein (A) and (B) can be performed consecutively in any order or simultaneously;
- and
- (C) comparing the elution time profiles from (A) and (B) to characterize complex types.
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Dependencies are inferred from the claim text.