MSACL 2023 Abstract

INTRODUCTION:
The pace of therapeutic peptide drug development has significantly increased over the last decade. Fueled by technological advancements in structural biology, recombinants and new synthetic and analytical techniques have shortened the time to market and increased the complexity of the peptide therapeutic. Peptides, while demonstrating several advantages over small molecules and larger biologics, including low immunogenicity, high specificity, and low cost, do have drawbacks, most notably stability and half-life. To improve peptide therapeutic biological function and pharmacokinetic profiles, several strategies have been employed, including, amino acid substitution, incorporation of unnatural amino acids, amino acid modifications, and cyclization. However, through the synthesis process, impurities can result that are often difficult to remove by purification alone. Therefore, robust analytical methods for analyzing peptide impurities are required. Hyphenated approaches such as high-resolution ion mobility mass spectrometry (HRIM-MS) have recently emerged as valuable tools for peptide therapeutic impurity analysis. HRIM is an analytical separation performed in the gas phase where ions are drawn through an inert background gas by an electric field. The mobility for these ions is dependent on their charge and shape and is measured as arrival time. In this way, challenging separations, like the separation of peptide isobars and isomers, have been demonstrated.

OBJECTIVES:
The main goal of this work was to leverage a combination of LC, High-Resolution Ion Mobility (HRIM), and QTOF approach to tackle the existing separation challenges of peptide isobaric and isomeric mixtures.

METHODS:
We used the MOBILion HRIM system based on Structures for Lossless Ion Manipulation (SLIM) to assess several types of isobaric and isomeric peptide modifications. Data acquisition was carried out on a high-resolution ion mobility (HRIM) MOBIE™ instrument (MOBILion Systems) coupled to a 6545XT QTOF (Agilent Technologies) with a 1290 Infinity II Autosampler (Agilent Technologies), using a combination of flow injection analysis (FIA) and reversed-phase liquid chromatography (RPLC) separation of standard peptides. For RPLC analyses of deamidated peptides, an Agilent Zorbax Eclipse C18, 50 x 2.1mm, 1.8 µm column at a flow rate of 0.4 mL/min was employed. All FIA and RPLC analyses were conducted using 0.1% formic acid in water and acetonitrile as mobile phases A and B, respectively, and the Agilent Dual Spray Jet Stream ESI Source operated in positive ion mode. The SLIM chamber was maintained at 2.5 Torr throughout the experiments. The SLIM-based HRIM Device was tuned and calibrated to optimize the transmission of ions between the mass range of m/z 300 and 3200. A series of experiments were carried out to determine the optimum traveling wave parameters required to trap and release ions to obtain the maximum resolution and sensitivity. Accurate mass, isotope spacing, ion mobility arrival time distribution, and Collision Cross Section (CCS determination were used for the identification of modified/unmodified peptide mixtures. Data processing, analysis, relative quantification, and visualization were achieved using HRIM Data Processor V1.10.29.1 (HRIM-DP), PNNL Preprocessor Version 4.0 (2022.02.17), Agilent IM-MS Browser.

RESULTS:
Herein we report our use of the MOBIE® HRIM from MOBILion with an Agilent LC-QTOF system to improve therapeutic peptide impurity analysis and demonstrate its applicability for resolving isobaric and isomeric peptide mixtures based on the gas phase separation of D- and L-amino acid isomers and deamidation and isomerization isomers. The novel HRIM method was first qualified using a range of peptide standards. We then evaluated the utility of the LC-HRIM-MS method for quantifying peptide impurities and compared LC-based quantification to quantification in the ion mobility domain. We demonstrate that implementing HRIM into the peptide analysis workflow enables confident separation, detection, and relative quantification of difficult-to-analyze isobaric and isomeric peptides reproducibly thereby eliminating the reliance on MS/MS fragmentation pattern. MOBIE® solves the 0.984 Da mass shift problem by providing an orthogonal separation, reducing the dependency on RPLC separation while maintaining confident identification and quantification of peptide modifications within isobaric/isomeric peptides.

CONCLUSION:
Mass spectrometry has proven extremely useful for analyzing amino acid modifications, facilitating mass determination, and sequencing of peptides. However, numerous challenges persist, including analyzing isobaric and isomeric peptide modifications, where existing separation technology has been limited in its utility. The LC-HRIM-QTOF system used is a powerful combination that can be implemented to enhance existing LC workflows for deeper sample characterization, reduce LC reliance to boost throughput, and add CCS values to existing compound identification.