Indirect homogeneous mobility shift assays for the detection of biologics in patient samples
Inventors
Salbato, Jared • Westin, Stefan • Chi-Kwan Ling, Nicholas • Jain, Anjali • Singh, Sharat
Assignees
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Publication Number
US-10422807-B2
Publication Date
2019-09-24
Expiration Date
Abstract
The present invention provides a sensitive and specific indirect homogeneous mobility shift assay using size exclusion chromatography to measure biologics such as vedolizumab and ustekinumab in a patient sample. The assays of the present invention are particularly advantageous for detecting the presence or level of biologics that target complex or large antigens including cell surface proteins, transmembrane proteins, heavily glycosylated proteins, and multimeric proteins, as well as antigens that cannot be purified, impure antigens, and partially or substantially purified antigens. The present invention also provides isolated soluble α4β7 integrin heterodimers and isolated soluble IL-12p40 monomers that are suitable for use in the indirect assays described herein.
Core Innovation
The invention provides a method for determining the presence or level of a biologic in a sample by using an indirect homogeneous mobility shift assay based on antigen-biologic complex formation and separation by size exclusion chromatography. An unlabeled soluble antigen is contacted with the sample to form an unlabeled complex between the antigen and the biologic in the sample, and a labeled form of the biologic is then contacted with the sample to form a labeled complex between the antigen and the labeled biologic.
The unlabeled and labeled complexes are subjected to size exclusion chromatography to separate the unlabeled and labeled complexes from free labeled biologic, and an amount of the free labeled biologic is detected. The detected amount of the free labeled biologic is compared to a standard curve of known amounts of the biologic, thereby determining the presence or level of the biologic in the sample. The approach is described as quantifying large and complex biologic targets in patient samples.
The disclosed soluble antigens include isolated soluble α4β27 integrin heterodimers and soluble IL-12p40 monomers, including IL-12p40 variants meeting sequence identity requirements to specified SEQ ID references. The document further addresses embodiments for assay validation, including sensitivity and quantitative performance metrics, and provides rationale for using soluble antigens that are difficult to purify or form multimers.
Claims Coverage
The disclosed claims center on one independent method claim that determines presence or level of a biologic by sequential antigen-complex formation with unlabeled soluble antigen followed by labeled biologic, separation by size exclusion chromatography, and quantification of free labeled biologic against a standard curve. Dependent claim coverage further specifies antigen composition and sequence-identity constraints, as well as particular biologics and standard-curve readout and analysis.
Unlabeled soluble antigen complex formation followed by labeled biologic complex formation
Contacting the sample with an unlabeled soluble antigen that binds to the biologic to form an unlabeled complex between the antigen and the biologic in the sample; contacting the sample from step (a) with a labeled form of the biologic to form a labeled complex between the antigen and the labeled biologic.
Size exclusion chromatography separation and detection of free labeled biologic
Subjecting the unlabeled and labeled complexes to size exclusion chromatography to separate the unlabeled and labeled complexes from free labeled biologic and to detect an amount of the free labeled biologic.
Standard curve comparison of detected free labeled biologic
Comparing the amount of the free labeled biologic detected in step (c) to a standard curve of known amounts of the biologic, thereby determining the presence or level of the biologic in the sample.
Soluble antigen as a soluble fragment, variant, monomer, or other specified protein form
Using an antigen comprising a soluble fragment, variant, or monomer of a membrane-bound protein, a glycosylated protein, a multimeric protein, an insoluble protein, and/or a large protein.
Soluble α4β27 integrin antigen
Using an α4β27 integrin.
Soluble fragment including α4 and/or β27 fragments with sequence identity constraints
A soluble fragment that includes an α4 fragment and/or a β27 fragment with amino acid sequences having at least 80% identity to specified SEQ ID NOs.
Use of ustekinumab as the biologic
Performing the method using the biologic ustekinumab.
AUC versus log standard-curve readout with interpolation
Calculating the biologic level by plotting an area under the curve (AUC) of a free labeled biologic versus the log of known biologic amounts and interpolating from that curve.
Overall claim coverage implements an indirect antigen-biologic complex approach with sequential unlabeled soluble antigen and labeled biologic, separates free labeled biologic from complexes by size exclusion chromatography, and quantifies by comparison to a standard curve. Dependent features specify soluble antigen forms, including α4β27 integrin and IL-12p40 monomer-related sequence-identity constraints, specify ustekinumab as an example biologic, and refine standard-curve analysis using AUC versus log with interpolation.
Stated Advantages
Determines the presence or level of a biologic in a sample by detecting and quantifying free labeled biologic separated from antigen-biologic complexes.
Enables quantification of large and complex biologic targets in patient samples using soluble antigens.
Allows soluble antigen selection including soluble forms and sequence-identity-qualified fragments for targets that may be difficult to purify or form multimers.
Provides a quantitative standard-curve analysis approach using AUC versus log and interpolation.
Documented Applications
Quantifying large and complex biologic targets, notably vedolizumab and ustekinumab, in patient samples using an indirect homogeneous mobility shift assay with size exclusion chromatography.
Performing the method for determining the presence or level of a biologic in a sample using soluble antigens including α4β27 integrin heterodimers and soluble IL-12p40 monomers.
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