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Publication Number
US-11543416-B2
Publication Date
2023-01-03
Expiration Date
Abstract
The present invention relates to a method for quantifying a therapeutic antibody in a sample of a human individual comprising a step of adding to a test sample which may contain therapeutic antibodies to be quantified a known amount of two or more labeled forms of said therapeutic antibodies.
Core Innovation
The invention provides a method for quantifying a therapeutic antibody in a sample of a human individual by mass spectrometric analysis after proteolysis. Two or more labeled forms of the therapeutic antibodies are added to a test sample containing therapeutic antibodies to be quantified, wherein the labeled forms are whole antibodies, and a pre-proteolysis sample is provided.
The sample is subjected to enzyme proteolysis to provide a proteolysis sample comprising proteolysis labeled peptides derived from the labeled whole therapeutic antibodies and proteolysis peptides derived from the therapeutic antibody in the test sample. The method determines the ratio between one or more selected proteolysis labeled peptides and one or more corresponding proteolysis peptides derived from the therapeutic antibody.
From the ratio determined, the amount of the therapeutic antibody in the test sample is calculated. The workflow uses corresponding labeled and non-labeled peptides generated from the whole labeled antibodies, and includes therapeutic antibodies such as anti-TNF antibodies and anti-cancer antibodies.
Claims Coverage
The claims center on one independent quantifying method, with dependent claims refining the scope through proteolysis conditions, protease selection, peptide selection, and sample preparation. Across the claim set, the inventive features emphasize whole-antibody labeled internal standards, enzyme proteolysis, mass spectrometric peptide-ratio measurement, and calculation of therapeutic antibody amount from those ratios.
Whole-antibody labeled forms as internal standards
Adding to a test sample containing therapeutic antibodies to be quantified a known amount of two or more labeled forms of said therapeutic antibodies, wherein the labeled forms are whole antibodies, to provide a pre-proteolysis sample.
Enzyme proteolysis generating labeled and endogenous peptides
Subjecting the pre-proteolysis sample to an enzyme proteolysis to provide a proteolysis sample comprising proteolysis labeled peptides derived from the labeled whole therapeutic antibodies and proteolysis peptides derived from the therapeutic antibody contained in the test sample.
Mass spectrometric ratio of selected corresponding peptides
Determining by mass spectrometric analysis the ratio between one or more selected proteolysis labeled peptides and one or more corresponding proteolysis peptides derived from the said therapeutic antibody.
Calculating antibody amount from the peptide ratio
Calculating from the ratio determined the amount of the said therapeutic antibody in the test sample.
Drug- and sequence-specific surrogate peptide selection
Selecting proteolysis labeled peptides chosen from a set of labeled peptide sequences corresponding to anti-TNF antibodies and anti-cancer antibodies via the listed SEQ ID NOs to enable the ratio determination.
Denaturing and non-denaturing proteolysis conditions
Defining enzyme proteolysis such that proteolysis includes both proteolysis performed under denaturing conditions and proteolysis performed under non-denaturing conditions.
Hinge-targeting protease selection including ideS
Using a hinge-targeting protease selected from a specified group including an Immunoglobulin-degrading enzyme from Streptococcus (ideS) as part of the proteolysis.
Anti-TNF antibody spiking and enrichment
Adding a known amount of two or more labeled anti-TNF therapeutic antibody forms to a test sample to provide a non-concentrated pre-proteolysis sample, and enriching the non-concentrated pre-proteolysis sample in the therapeutic antibodies to provide a pre-proteolysis sample.
Overall, the claims cover a mass spectrometry-based quantification method that uses two or more labeled whole therapeutic antibodies as internal standards, generates labeled and endogenous peptides via enzyme proteolysis, measures the ratio between selected corresponding labeled and endogenous peptides, and calculates the therapeutic antibody amount from that ratio. Dependent claims further specify therapeutic targets and surrogate peptide SEQ ID ranges, proteolysis condition options, hinge-targeting protease selection including ideS, and an anti-TNF spiking and enrichment workflow.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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