FIELD OF THE INVENTION

The invention is generally related to systems and methods for improved protein sequencing.

BACKGROUND OF THE INVENTION

Confirmation of the primary sequence of therapeutic monoclonal antibodies (mAbs) by liquid chromatography-mass spectrometry (LC-MS) analysis after tryptic digestion is an essential assay routinely performed in the biotechnology industry. Complete sequence coverage is highly desirable for peptide mapping analysis of therapeutic monoclonal antibodies, but it is not always achievable by tryptic digestion alone. Small and/or hydrophobic peptides with poor or no retention on the commonly used C18 column often miss MS detection, resulting in gaps in the coverage map. It is particularly problematic if the missed sequence belongs to critical complementarity-determining regions. To achieve 100% coverage, a secondary enzymatic digestion is frequently required. Alternatively, the emerging capillary zone electrophoresis coupled to mass spec (CE-MS) technology can achieve 100% coverage, but availability of such platforms is still limited.

Therefore, it is an object of the invention to provide systems and methods for obtaining complete amino acid sequences of proteins.

SUMMARY OF THE INVENTION

A dual-column liquid chromatograph-mass spectrometry (LC-MS) system and methods of use thereof are provided. One embodiment provides a dual column liquid chromatography system including 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. The system can optionally include a detector, for example a UV detector and/or a mass spectrometer, and a collection vessel. Typically, the first column is packed with material for separating non-polar and hydrophobic peptides and the second column is packed with material for separating polar peptides. For example, the first column can be a C18 column and the second column can be a porous graphitic column.

In another embodiment the multi-port switching diverter valves are configured to allow fluid from the first column to flow through the second column. In still another embodiment, the multi-port switching diverter valves are configured to allow fluid from the first column to bypass the second column and flow to a mass spectrometer.

Another embodiment provides a method for determining the sequence of a protein by digesting the protein to produce peptide fragments of the protein, 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. 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. The method includes eluting the peptides retained on the first column in a way in which the eluted peptides bypass the second column. The eluted peptides are analyzed by mass spectrometry to determine the amino acid sequence of the peptides. Peptides from the second column are also eluted and analyzed by mass spectrometry to determine the amino acid sequence of the eluted peptides. The combined sequences of the eluted peptides from the first and second columns provide a complete amino acid sequence of the protein. In one embodiment, the protein being sequenced is an antibody, for example a monoclonal antibody, or a fusion protein.

In another embodiment the first column is packed with material for separating non-polar peptides and the second column is packed with material for separating polar peptides. For example, the first column can be a C18 column and the second column can be a porous graphitic column. In certain embodiments, the protein is reduced and alkylated prior to digestion. The proteins are typically enzymatically digested, for example using trypsin.

In one embodiment, peptides eluted from the C18 column are analyzed by mass spectrometry at a scan range of 300 to 2000 m/z and peptides eluted from the PGC column is analyzed by mass spectrometry at a scan range of 200 to 2000 m/z.

