Multidimensional LC System for Antibody Peptide Mapping
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Solution Overview
Problem
Current automated systems for peptide mapping in biopharmaceutical analysis are limited by the use of digestion columns that cannot tolerate high pressures, restricting the use of ultrahigh performance (UHPLC) columns and resulting in reduced sequence coverage, particularly in the detection of small, polar peptides.
Innovation Solution
A multidimensional liquid chromatography (LC) system that decouples the digestion and separation modules, allowing the use of UHPLC columns by preventing backpressure and incorporating a trapping module for solvent adjustment, thereby enabling the detection of small polar peptides and increasing sequence coverage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If digestion columns are used in automated LC systems, then sample processing can be automated, but the system pressure is limited to approximately 170 bar, preventing the use of UHPLC columns
Solution Approach 1:
The system is divided into distinct functional modules: a first LC system for digestion (low pressure tolerant) and a second LC system for peptide mapping (high pressure UHPLC). This segmentation allows each module to operate at its optimal pressure independently, resolving the contradiction between automation and pressure limits.
Solution Approach 2:
A quadrupole mass filter is introduced as an intermediary between the two LC systems. It receives peptides from the first system and filters/selects them before introducing them to the second UHPLC system, enabling the connection between low-pressure digestion and high-pressure mapping without direct pressure transmission.
2Extent of automation
If standard digestion columns are used, then automated digestion can be performed, but backpressure from downstream processes damages the digestion columns
Solution Approach 1:
The system separates digestion and peptide mapping into independent LC systems. The first system performs digestion at low pressure, while the second system performs mapping at high pressure. This segmentation eliminates backpressure transmission from the mapping stage to the digestion stage, protecting the digestion columns.
Solution Approach 2:
The quadrupole mass filter acts as a pressure-isolating intermediary between the two systems. It decouples the pressure regimes, allowing the digestion system to operate at low pressure while the mapping system operates at high pressure without transmitting harmful backpressure to the digestion columns.
3Measurement precision
If UHPLC columns are used for peptide mapping, then sequence coverage and detection of small polar peptides is improved, but the system requires high pressure compatibility throughout the entire LC path
Solution Approach 1:
The LC path is segmented into two independent systems: the first for digestion (pressure-independent) and the second for peptide mapping (high pressure UHPLC). This allows UHPLC columns to be used for mapping where high pressure and sequence coverage are beneficial, without requiring the entire system to be pressure-compatible.
Solution Approach 2:
The quadrupole mass filter serves as an intermediary that enables the use of UHPLC columns by filtering and preparing peptides from the first system before introducing them to the second UHPLC system. This intermediary allows the high-pressure mapping capability to be isolated to only the necessary portion of the system.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The system facilitates the use of UHPLC columns up to 1300 bar, enhancing peptide mapping analysis by improving sequence coverage and retaining small polar peptides, which is crucial for ensuring the safety and efficacy of biopharmaceutical products.
Implementation Method 1
a digestion column containing an immobilized proteolytic enzyme
Implementation Method 2
a trapping column for holding the sample after digestion in the digestion module
Implementation Method 3
a separation column for separating analytes in the sample after release of the sample from the trapping column
Data Source
AI summary
The present application is directed to multidimensional liquid chromatography (LC) systems. In certain aspects, provided is a multidimensional LC system comprising a digestion module having a digestion column containing an immobilized proteolytic enzyme, a trapping module having a trapping column for holding the sample after digestion in the digestion module, and a separation module having a separation column for separating analytes in the sample after release of the sample from the trapping column. In certain aspects, in the direction of flow through the LC systems described herein, the modules are in the following order: digestion module; trapping module, and then separation module.