EA044768B1 - TWO-COLUMN LC-MS SYSTEM AND METHODS OF ITS APPLICATION - Google Patents

Description

Field of invention

The invention generally relates to systems and methods for improving protein sequencing.

BACKGROUND OF THE INVENTION

Confirmation of the primary sequence of therapeutic monoclonal antibodies (mAbs/mAbs) by liquid chromatography-mass spectrometry (LC-MS) after tryptic digestion is an important analysis commonly performed in the biotechnology industry. Complete sequence coverage is highly desirable for peptide mapping analysis of therapeutic monoclonal antibodies, but is not always achievable using tryptic digestion alone. Small and/or hydrophobic peptides with poor or no retention on the commonly used C18 column are often not detected by MS, resulting in gaps in the coverage map. This is especially problematic if the missing sequence belongs to critical regions that determine complementarity. Secondary enzymatic digestion is often required to achieve 100% coverage. Alternatively, the new technology of capillary zone ectrophoresis coupled to mass spectrometry (CE-MS/CE-MS) can provide 100% coverage, but the availability of such platforms is still limited.

It is therefore an object of this invention to provide systems and methods for obtaining complete amino acid sequences of proteins.

Brief description of the invention

A two-column liquid chromatography-mass spectrometry (LC-MS) system and methods for its application are proposed. In one embodiment, a two-column liquid chromatography system is provided, including a first fluid communication column with a first multi-port switching diverter valve, a second fluid communication column with a first multi-port switching diverter valve; a second multiport switching redirect valve in fluid connection with the first multiport switching redirection valve; a mass spectrometer in fluid connection with a second multiport switching redirection valve; a collection reservoir in fluid connection with a second multiport switching redirect valve; and a computer in electronic communication with the two-column chromatography system for controlling the flow of liquid through the system by configuring the ports of the first and second multiport switching redirect valve into multiple configurations. Said system may further comprise a detector, for example a UV detector and/or a mass spectrometer, and a collection reservoir. Typically, the first column is filled with material to separate non-polar and hydrophobic peptides, and the second column is filled with material to separate polar peptides. For example, the first column may be a C18 column and the second column may be a porous graphite column.

In another embodiment, the multiport switching redirect valves are configured to allow fluid from a first column to pass through a second column. In yet another embodiment, the multiport switching redirect valves are configured to allow liquid from the first column to bypass the second column and flow into the mass spectrometer.

In another embodiment, a method is provided 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. . Peptides not retained in the first column are passed directly to a second high performance liquid chromatography column, wherein at least a portion of the peptides not retained in the first column are retained in the second column. The method includes eluting peptides retained in the first column such that 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 the complete amino acid sequence of the protein. In one embodiment, the protein being sequenced is an antibody, such as a monoclonal antibody or fusion protein.

In another embodiment, the first column is filled with material for separating non-polar peptides and the second column is filled with material for separating polar peptides. For example, the first column may be a C18 column and the second column may be a porous graphite column. In certain embodiments, the protein is reduced and alkylated before cleavage. Proteins are usually digested enzymatically, for example using trypsin.

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

- 1 044768 are analyzed by mass spectrometry in the scanning range from 200 to 2000 m/z.

Brief description of graphic materials

Fig. 1 is a diagram of an exemplary two-column LC-MS system.

Fig. 2A-2C are schematic diagrams of the configuration of dual redirection valves. The lines indicate the solvent flow path when the redirect valves are configured in Load, C18 Elution, or PGC Elution modes. The 1-x/1-x position refers to the redirect valve port positions 1 and 2 respectively.

Fig. 3 is a user interface image showing an exemplary mass spectrometer acquisition method setup for a two-column PGC-C18 configuration.

Fig. 4 is a table showing a representative example of a C18-PGC elution gradient.

In fig. 5A and 5B are photographs of a first multiport switching redirect valve located above a second multiport switching redirection valve.

Fig. 6A is a screenshot of a user interface showing parameters for C18 elution. Fig. 6B is a screenshot of a user interface showing parameters for PGC elution.

Fig. 7A,B are an exemplary total ion chromatogram (TIC) of a C18-PGC peptide mapping analysis.

Detailed description of the invention

Definitions.

As used herein, the term peptide mapping refers to a technique for characterizing proteins and elucidating their primary amino acid structures. It is a widely used method for characterizing monoclonal antibodies and other recombinant protein-based pharmaceuticals.

As used herein, the term tryptic peptides refers to peptides that are generated by digestion of larger proteins into smaller peptides using the serine protease trypsin. Trypsin cleaves proteins into peptides with an average size of 700 to 1500 Daltons by hydrolyzing peptide bonds on the carboxy-terminal side of the amino acid residues arginine and lysine.

As used herein, the term porous graphitic carbon (PGC) column refers to a chromatography column that is composed of porous graphitic carbon. It is a good stationary phase in liquid chromatography due to its unique retention and separation of highly polar compounds.

As used herein, the term C18 column refers to a reverse phase chromatography column commonly used in liquid chromatography. C18 columns consist of carbon chains linked to silica particles inside the column. These columns are typically used to separate hydrophobic and nonpolar molecules that bind to the carbon chains of silica.

As used herein, the term liquid chromatography (LC) refers to a technique used to separate, identify, and quantify components in a mixture. In column liquid chromatography, a liquid mobile phase passes through a column and components of the mobile phase interact with a solid stationary phase. The composition of the mobile phase can be changed during the separation cycle to change the strength of interaction of the compounds of interest. As the mobile phase continues to flow through the column, the eluent is usually collected in fractions, monitoring the concentrations of compounds eluting from the column over time to produce an elution curve or chromatogram.

Liquid chromatography is typically coupled with mass spectrometry to continuously measure protein elution from a column by measuring the light absorbance at 280 nm of the amino acid tryptophan. Each individual peak in the chromatogram represents a unique component separated by the column, and the area under the peak corresponds to the amount of that compound eluted from the column. A single peak may contain multiple proteins, so further analysis of the eluted fractions is often required.

As used herein, the term stationary phase refers to the substance that remains fixed in the column. The most commonly used stationary phase columns are carbon-chain silica, phenyl-linked silica, and cyano-linked silica.

As used herein, mobile phase and liquid phase can be used interchangeably and refer to mixtures of water or aqueous buffers and organic solvents that are used to elute compounds from columns. The most common mobile phase solvents include, but are not limited to, acetonitrile, methanol, tert-hydrofuran, ethanol, or isopropyl alcohol.

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As used herein, the terms elution, elute, and eluted refer to the process of removing one material from another by washing with a solvent.

As used herein, the term total ion chromatogram (TIC) is a type of chromatogram created by summing the intensities of all mass spectral peaks belonging to the same scan. The TIC includes background noise as well as sample components.

As used herein, the term TFA is an abbreviation for trifluoroacetic acid, a commonly used modifier for the separation of peptides in reverse phase liquid chromatography. Modifiers are substances added to the mobile phase that interact with both the stationary phase and the sample to alter retention.

The term LC-MS refers to an analytical chemistry technique that combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS).

Two-column LC-MS system.

In a standard peptide mapping configuration, peptides are retained on a C18 column installed in the LC system and eluted onto the mass spectrometer using a single 6-port switch valve that switches between direct LC flow to the mass spectrometer or to a waste reservoir.

One embodiment provides a two-column liquid chromatography system, including a first column in fluid communication with a first multiport switching redirect valve; a second column in fluid communication with the first multiport switching redirect valve; a second multiport switching redirection valve that is in fluid communication with the first multiport switching redirection valve; a mass spectrometer that is in fluid communication with a second multiport switching redirect valve; a collection reservoir that is in fluid communication with a second multiport switching redirect valve; and a computer in electronic communication with the two-column chromatography system for controlling the flow of liquid through said system by configuring ports of the first and second multiport switching redirect valves in multiple configurations.

The system may further include a detector, such as a UV detector and/or a mass spectrometer, and a collection reservoir. Typically, the first column is filled with material for separating non-polar peptides, and the second column is filled with material for separating polar peptides. For example, the first column may be a C18 column and the second column may be a porous graphite column.

In another embodiment, the multi-port switching redirect valves are configured to pass fluid from the first column through the second column. In yet another embodiment, multiport switching redirect valves are configured to allow liquid from the first column to bypass the second column and flow to the mass spectrometer.

Fig. 1 is a diagram of an exemplary system 100. The system 100 includes a column 101. Column 101 is typically an HPLC column packed with material for separating non-polar and/or hydrophobic peptides. Column 101 is in fluid communication with multiport switching redirect valve 103. System 100 also includes a second column 102 also in fluid communication with multiport switching redirect valve 103. Multiport 103 is in fluid communication with second multiport switching redirect valve 104 Multiport switching redirect valve 104 is in fluid communication with mass spectrometer 106 and collection reservoir 105.

Double redirection valves.

An exemplary configuration of two switching redirection valves is shown in FIG. 2A-2C. In fig. 5A and 5B are photographs of illustrative dual redirection valves. The first position of the redirect valve is called 1-6, in which solvent flow enters through port 1 and exits through port 6. The second position of the redirect valve is called 1-2, in which solvent flow enters through port 1 and exits through port 2. Position 1-x /1-x refers to the port positions of redirect valves 1 and 2 respectively. Therefore, for example, 1-6/1-6 indicates that LC solvent enters port 1 and exits port 6 of redirect valve 1, then enters port 1 and exits port 6 of redirect valve 2. The first redirect valve is set so that that position 1-6 will direct the flow from the LC through the PGC column to the inlet of the second redirect valve. Connections to the PGC column are located at ports 6 and 3 of redirect valve 1, as shown in FIG. 2A. More specifically, the output line from the LC is connected to port 1 of redirect valve 1, the PGC column is connected at ports 3 and 6 with flow direction from port 6 to port 3, and the output line from port 2 of redirect valve 1, which is connected to port 1 of diverter valve 2. The second diverter valve is set such that positions 1-6 direct flow from the diverter valve 1 outlet port to the mass spectrometer, and positions 1-2 redirect flow to waste. More specifically, the output of redirect valve 1 is connected to port 1, the output of redirect valve 2 connects port 2 to the mass spectrometer, and any output connects port 6 to the waste reservoir. Peptides can be loaded onto either a C18 column or a PGC column at the 1-6/1-6 position.

Elution of tryptic peptides from the C18 column will occur at the 1-2/1-2 position as described in FIG. 2B. Peptides will be eluted from the PGC column at the 1-6/1-2 position as described in FIG. 2C.

In one embodiment, the flow rate through a two-column LC-MS system may be 0.25 ml/min. One skilled in the art will appreciate that the disclosed systems may use other flow rates than those used in typical LC-MS.

In one embodiment, the mobile phase is 0.05% TFA in water for loading and 0.045% TFA in acetonitrile for the elution phase. A typical elution gradient is shown in FIG. 4.

In one embodiment, 0.1% formic acid (FA) was used for loading and 0.1% formic acid in acetonitrile was used for elution.

Switching the redirect valve positions can be controlled in the mass spectrometer settings, as demonstrated in FIG. 3. The switching time of the switches correlates with the C18-PGC LC gradient, an example of which is demonstrated in FIG. 3

Elution of peptides.

In one embodiment, proteins are sequenced using a two-column LC-MS system disclosed herein. Proteins are typically reduced and alkylated using TCEP and iodoacetamide and then digested with trypsin to produce tryptic peptides. Loading and elution of tryptic peptides from a C18 column and a PGC column can be achieved sequentially. For example, trypsinized mixtures are injected into LC-MS and then loaded onto C18 and PGC columns with diverter valves set to 1-6/1-6. Tryptic peptides are first stored in column C18. Hydrophilic peptides that are not retained in the C18 column are then eluted into the PGC column below and remain in the PGC column. Fig. 6A and 6B show exemplary elution parameters. Following the loading step, the C18 column first elutes the peptides with the redirect valves set to bypass the PGC column using the 1-2/1-2 position. After completion of elution on the C18 column, the gradient equilibrates to the initial conditions. PGC column elution occurs by diverting flow through the PGC column using redirection valves in the 1-6/1-2 position. C18 elution must occur while bypassing the downstream PGC column to avoid clogging the PGC column with larger tryptic peptides remaining on the C18 column or interference signal from small hydrophilic peptides remaining on the PGC column.

Mass spectrometric analysis.

The peptide eluate from the C18 and PGC columns enters the mass spectrometer for MS analysis. In one embodiment, the scan range for the C18 eluent is set to 300 to 2000 m/z to maintain a normal baseline TIC level. In one embodiment, the scan range for the PGC eluate is set to 200 to 2000 m/z to detect small peptides. The disclosed methods have been demonstrated to provide 100% sequence coverage for all molecules tested.

The two-column LC-MS system can be implemented with minor modifications to existing C18 peptide mapping schemes while providing greater sequence coverage and valuable information. Therefore, a two-column LC-MS system offers a simple solution to achieve complete mAb sequence coverage in a single LC-MS analysis.

Examples

Example 1: Sequence coverage analysis.

Ways.

Monoclonal antibody samples were first reduced and alkylated using TCEP and iodoacetamide before trypsin digestion. The digestion mixtures were then injected into a two-column LC-MS system consisting of a C18 column and a PGC column. During the loading step, tryptic peptides were first retained on a C18 column. Hydrophilic peptides not retained in this column were eluted and retained in the underlying PGC column. Elution first occurred on the C18 column while a redirect valve allowed flow to bypass the PGC column. After elution on the C18 column was completed and the gradient had equilibrated to initial conditions, the elution of peptides on the PGC column was started by diverting the flow through the PGC column.

Results.

It was observed that the common peptides remaining on the PGC column, but not on the C18 column, were dipeptides containing a C-terminal Lys or Arg. Using the exact masses of MS1 and characteristic properties

Claims (12)

ammonium ion signs during HCD fragmentation, the identity of these short peptides can be confidently confirmed. Using the NIST mAh reference standard and several proprietary NIST mAh molecules, it was demonstrated that the disclosed method could provide 100% sequence coverage for all molecules tested. Fig. 7 shows a total ion chromatogram of an exemplary protein that has been sequenced. CLAIM 1. A method for determining the sequence of a protein, comprising: protein cleavage to produce peptide fragments; loading peptide fragments onto a first high performance liquid chromatography column under conditions in which some of the peptide fragments are retained in the first column and some of the peptide fragments are not retained in the first column, wherein peptides that are not retained in the first column flow directly into the second column for high performance liquid chromatography, wherein at least a portion of the peptides not remaining in the first column remain in the second column; eluting peptides retained in the first column, the eluted peptides bypassing the second column, and analyzing the eluted peptides using mass spectrometry to determine the amino acid sequence of the peptides; and eluting the peptides from the second column and analyzing the eluted peptides using 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 the complete amino acid sequence of the protein. 2. The method of claim 1, wherein the protein is an antibody or a fusion protein. 3. The method of claim 2, wherein the antibody is a monoclonal antibody. 4. The method according to any one of claims 1 to 3, in which the first column is filled with material for separating non-polar peptides, and the second column is filled with material for separating polar peptides. 5. The method according to any one of claims 1 to 4, wherein the first column is a C18 column. 6. The method according to any one of claims 1 to 5, wherein the second column is a porous graphite column. 7. Method according to any one of claims 1 to 6, wherein the protein is reduced and alkylated before cleavage. 8. Method according to any one of claims 1 to 7, in which the protein is digested with trypsin. 9. The method according to claim 5, in which the peptide eluted from the C18 column is analyzed by mass spectrometry in the scanning range from 300 to 2000 m/z. 10. The method of claim 6, wherein the peptides eluted from the PGC column are analyzed by mass spectrometry over a scanning range of 200 to 2000 m/z. 11. Two-column liquid chromatography system for determining the amino acid sequence of a protein, containing: a first high performance liquid chromatography column in fluid communication with a first multiport switching redirect valve where peptide fragments are loaded onto the first column; a second high-performance liquid chromatography column in fluid communication with the first multiport switching redirect valve, wherein a portion of the peptide fragments not retained in the first column flows directly into and is retained in the second column, thereby eluting the peptide fragments, held in the first column and peptide fragments held in the second column; a second multiport switching redirect valve in fluid communication with the first multiport switching redirection valve; a mass spectrometer in fluid communication with a second multiport switching redirect valve; a collection reservoir in fluid communication with a second multiport switching redirect valve, wherein the eluted peptide fragments from the first column and the eluted peptide fragments from the second column are analyzed by a mass spectrometer to determine the amino acid sequence of the peptide fragments from the first column and the second column, respectively, at in this case, the combination of the sequence of eluted peptides from the first column and from the second column provides the complete amino acid sequence of the protein; and a computer in electronic communication with the two-column chromatography system for controlling the flow of liquid through the system by configuring ports of the first and second multiport switching control valves in multiple configurations. 12. The system according to claim 11, further comprising a mass spectrometer communicated using -