Heavy peptide approach to accurately measure unprocessed c-terminal lysine in antibodies
AssigneeRegeneron Pharmaceuticals, Inc.
InventorsTyler Greer, Milos Cejkov, Reid O'Brien Johnson, Xiaojing Zheng, Ning Li
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Abstract
The present disclosure provides a method for measuring post-translational modifications in proteins such as antibodies. In particular, the method may be used to quantify C-terminal truncation in antibodies that incorporates heavy isotopic standards for both the unprocessed C-terminal K peptide and the truncated C-terminal K peptide to build a calibration curve and quantify this PTM using mass spectrometry. Quantification of post-translational modifications may occur in a single liquid chromatography tandem mass spectrometry (LC-MS2) run.
Claims16
A method for quantifying unprocessed C-terminal lysine in a peptide (K peptide), comprising:
- mixing a set of heavy C-terminal peptide standards with a peptide digest;
- generating a calibration curve of a peptide signal of the unprocessed C-terminal K response relative to that of a truncated (des-K) peptide;
- and
- analyzing and quantifying the percentage of K peptide using liquid chromatography mass spectrometry.
A method for quantifying unprocessed C-terminal lysine in a peptide (K peptide), comprising:
- digesting a protein with a protease to produce a peptide digest;
- mixing the peptide digest with a set of heavy C-terminal peptide standards, wherein the set of heavy C-terminal peptide standards comprises equimolar concentrations of Δ4 K peptide:Δ4 des-K peptide, Δ8 K peptide:Δ4 des-K peptide, Δ12 K peptide:Δ4 des-K peptide, and Δ16 K peptide:Δ4 des-K peptide;
- generating a calibration curve of a peptide signal of the unprocessed C-terminal K response relative to that of a truncated (des-K) peptide;
- and
- analyzing and quantifying the percentage of K peptide using liquid chromatography mass spectrometry.
A kit for quantifying unprocessed C-terminal lysine in a peptide (K peptide), comprising:
- des-K peptide standards;
- K peptide standards;
- heavy des-K-peptide standards;
- heavy K-peptide standards;
- and
- instructions for use.
Dependencies are inferred from the claim text.