A multidimensional lc system for analysing antibodies
AssigneeHoffmann-La Roche Inc.
InventorsAnja BATHKE, Sina Nadine HOELTERHOFF, Saban OEZIPEK
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Abstract
The invention related to a multidimensional LC system for analysing antibodies. The system provides multiple different measurement samples from a single sample, for example, the system may provide a native antibody sample for measurement, a reduced antibody sample for measurement and digested sample for peptide mapping from a single sample. The system has a fractionation module, a reduction module, a digestion module, a separation module and at least a first and a second splitter for splitting the flow.
Claims15
A multidimensional liquid chromatography (LC) system comprising
- a fractionation module having a fractionation column for providing fractionated samples
- a first splitter fluidly connectable to and downstream from the fractionation module and fluidly connectable to and upstream from the reduction module wherein the first splitter has an inlet, a first outlet and a second outlet for splitting the fractionated samples into a first measurement sample, such as a native antibody measurement sample, that is passed through the first outlet and a sample for reduction that is passed through the second outlet
- a reduction module having a reduction column fluidly connectable to the second outlet of the first splitter
- a second splitter fluidly connectable to and downstream from the reduction module and fluidly connectable to and upstream from the digestion module wherein the second splitter has an inlet, a first outlet and a second outlet for splitting the reduced sample into a second measurement sample, such as a reduced antibody measurement sample, that is passed through the first outlet, and a sample for digestion that is passed through the second outlet
- a digestion module having a digestion column containing an immobilised proteolytic enzyme for digesting the reduced sample to provide a digested sample wherein the digestion column is fluidly connectable to the second outlet of the second splitter
- a separation module having a separation column for separating analytes and providing a third measurement sample, such as a peptide mapping sample.
The multidimensional LC system of any one of the preceding claims wherein the system has a first valve assembly configured so that in a first position:
- the inlet of the first splitter is fluidly connected to the fractionation module and, when present, the buffer exchange module or the multiple heart cutting valve;
- the first outlet of the first splitter is fluidly connected to another module of the system such that a first measurement sample can be split from the flow;
- and the second outlet of the first splitter is fluidly connected to the reduction module,
- and in a second position the inlet and the second outlet of the first splitter are connected in a loop and the reduction module is fluidly connected to the second splitter and when present the reduction pump.
The multidimensional LC system of any one of the preceding claims wherein the system has a second valve assembly configured so that in a first position the second outlet of the second splitter is fluidly connected to the digestion module and, when present, the trapping module and in a second position of the second valve assembly, the digestion module is isolated and is not in fluid connected with any other components.
The multidimensional LC system of any one of the preceding claims wherein the system has a fourth valve assembly configured so that in a first position the first outlet of the first splitter is fluidly connected to another module of the system and so that in a second position the first outlet of the second splitter is fluidly connectable to another module of the system optionally wherein the another module may be an analysis module such as a mass spectrometer.
The multidimensional LC system of any one of the preceding claims wherein the system has a third valve assembly configured so that the first outlet of the second splitter is connected to another module of the system when the third valve assembly is in the first position and the separation module is fluidly connected to another module of the system when the third valve assembly is in the second position, preferably in both cases the fourth valve assembly is in the second position.
The multidimensional LC system of any one of the preceding claims further comprising a multiple heart cutting valve fluidly connected to the fractionation column and is after the fractionation column in the direction of flow.
The multidimensional LC system of any one of the previous claims wherein the digestion module has a fourth splitter such as static mixer or a zero delay volume T-Piece downstream from digestion column in the direction of flow or a third splitter such as static mixer or a zero delay volume T-Piece upstream from the digestion column in the direction of flow.
The multidimensional LC system of any one of the previous claims further comprising an analysis module for analysing the first measurement sample, the second measurement sample and the third measurement sample optionally wherein the analysis module comprises a mass spectrometer (HRMS) or a Single Quad mass spectrometer;
- an evaporative light scattering detector (ELSD):
- a UV detector;
- or a diode array detector (DAD).
Dependencies are inferred from the claim text.