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US 11,435,359 B2Granted
Dual-column LC-MS system and methods of use thereof
AssigneeRegeneron Pharmaceuticals, Inc.
InventorShunhai Wang
Life of this patent
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Abstract
Methods for achieving complete sequence coverage of monoclonal antibodies by trypsin digestion and dual-column LC-MS system are provided. The disclosed method improves upon current techniques for standard peptide mapping.
Claims17
A method for determining the sequence of a protein comprising:
- reducing and alkylating the protein with tris(2-carboxyethyl)phosphine (TCEP) and iodoacetamide prior to digesting the protein to produce peptide fragments;
- loading the peptide fragments onto a first high performance liquid chromatography column under conditions in which some of the peptide fragments are retained on the first column and some of the peptide fragments are not retained on the first column, wherein the peptides not retained on the first column flow directly into a second high performance liquid chromatography column where at least a portion of the peptides not retained on the first column are retained on the second column;
- eluting the peptides retained on the first column, wherein the eluted peptides bypass the second column and analyzing the eluted peptides by a mass spectrometry to determine the amino acid sequence of the peptides;
- and
- eluting peptides from the second column and analyzing the eluted peptides by mass spectrometry to determine the amino acid sequence of the eluted peptides, wherein the combined sequences of the eluted peptides from the first and second columns provide a complete amino acid sequence of the protein.
2 more elements
A dual column liquid chromatography system comprising:
- a first column in fluid communication with a first multi-port switching diverter valve;
- a second column in fluid communication with the first multi-port switching diverter valve;
- a second multi-port switching diverter value in fluid communication with the first multi-port switching diverter valve;
- a mass spectrometer in fluid communication with the second multi-port switching diverter valve;
- a collection vessel in fluid communication with the second multi-port switching diverter valve;
- and
- a computer in electronic communication with the dual column chromatography system for controlling fluid flow through the system by configuring the ports of the first and second multi-port switching diverter valves in multiple configurations, wherein
- the protein is reduced and alkylated with tris(2-carboxyethyl)phosphine (TCEP) and iodoacetamide prior to digestion.
5 more elements
Dependencies are inferred from the claim text.