Accepted: Biopharm internal poster

Introduction

As biotherapeutics become more complex and companies strive for increased intellectual property protection, ever more sophisticated tools are being investigated to provide in-depth detailed molecular characterisation. Such studies focus on acquiring knowledge of the post-translational modifications (PTM’s) including glycosylation, oxidation and deamidation present in the protein product, with control of these being paramount. Mass spectrometry (MS) is a central technique in biopharmaceutical characterization due to its ability to report on such a wide range of attributes. However, the presence of isobaric PTM’s with differing biological properties can often be refractory to traditional LC-MS workflows even when chromatographically separated. In this work we investigate cyclic ion mobility technology as a means to distinguish isomeric PTM’s to improve biotherapeutic characterisation

Methods

Studies were performed on a cyclic ion mobility-enabled quadrupole time-of-flight (Q-cIM-oaToF) mass spectrometer. The circular geometry of the cIM device minimizes instrument footprint whilst providing a longer separation path (1m), Ions can cycle the path length multi-times, provides significantly higher resolution over a selected mobility range.. The multifunctional T-WAVE ion entry/exit array allows mobility selectivity by ejecting species within a range of mobilities,to a pre-store array, enabling multiple stages of ion mobility selection and separation (IMSn). The native and iso-aspartic variants of the synthetic peptide WGGDGFYAMDYWGQGTLVTVSSASTK (T12-D and T12-isoD, respectively) were used in this study to mimic deamidation products from protein biotherapeutics. The peptides were infused under nano-electrospray conditions in 97:2.9:0.1 Water/Acetonitrile/Formic acid at a concentration of 1 micromolar.

Preliminary Data

The two isomeric peptides T12-D and T12-isoD formed primarily the [M+3H]3+ ion at m/z 928. After a single pass of the cIM device it was observed from its arrival time distribution (ATD) that T12-D formed two conformers, one compact (1) and one more extended (2) with approximately 90 % of the signal present as conformer 1. By comparison T12-isoD was found to populate only conformer 2. These data suggest that the presence of the isoD variant biases the conformation of the T12 peptide to the more extended form under these conditions.
The T12 peptide was selected by the resolving quadrupole and subjected to fragmentation in the trap ion guide situated prior to the cIM device. This enables separation and structural analysis of the resulting product ions by ion mobility followed by mass analysis. In order to probe the product ions in detail we subjected the peptides to both multi-pass and IMSnstudies. Peptide product ions were initially separated over a single cIM pass (resolution approx. 65). A defined mobility range containing the product ions of interest was then selected by ejection to the pre-cIM storage ion guide, with all other ions outside of this range discarded. The stored ions were then reinjected into the cIM device for multiple passes to acquire at a higher mobility resolution. Upon comparing T12-D and T12-isoD product ions, several b-type ions showed differences in their ATD, indicating different gas phase structures. When conducting the experiments with an equimolar mixture of T12-D and T12-isoD the product ion ATD indicated, and was consistent with, a mixture of the two forms. These preliminary data indicate that multifunction cIM could be used to identify and aid in sequencing peptide isomers. Further data will be collected on a range of additional modified peptides with on-line LC separation for chromatographic separation.

Novel Aspect

IMSnfor distinguishing post-translational modifications in biopharmaceutical analysis

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A post-doc is presenting author on this abstract? No
A graduate student is presenting author on this abstract? No
An undergraduate student is presenting author on this abstract? No

Poster:

Ion Mobility: Applications

Submitting Author:

Jim Langridge
Waters Corporation
Wilmslow,
james_langridge@waters.com