TP038 Native Ion Mobility Mass Spectrometry for the Characterization of Biotherapeutics
Dale A. Cooper-Shepherd, Jakub Ujma, Kevin Giles, Laetitia F. Denbigh, Nick Tomczyk
Introduction
Biopharmaceutical products provide a complex analytical challenge to the modern pharma industry. Not only must laboratories characterize the primary structure of protein species, but also the tertiary and quarternary (higher order) structure. One method often used in research laboratories for higher order structure analysis is native ion mobility spectrometry-mass spectrometry (IMS-MS). Due to the expansion of the biopharmaceutical industry and the broadening range of drugs being investigated, instrumentation is required to carry out advanced experiments in a routine manner. We describe a method to carry out native IMS-MS on a routine high resolution LC-IMS-MS platform. We also describe investigations into the applicability of high resolution-IMS-MS (HR-IMS-MS) for biopharmaceutical analysis using a cyclic IMS-enabled research platform.
Methods
Size exclusion chromatography (SEC)-native ion mobility spectrometry-mass spectrometry was conducted on trastuzumab and the antibody-drug conjugate trastuzumab-emtansine (without deglycosylation). SEC was conducted on an ACQUITY I-class UPLC system with UPLC BEH200 SEC 4.6 x 150 mm, 1.7 µm column. 100 mM ammonium acetate was used as the mobile phase. SEC was conducted online with a VION IMS QTOF (Waters Corp., Wilmslow, UK). Source temperatures and voltages were optimized for the transmission of intact natively-folded protein ions. Data were processed using UNIFI software. Enhanced resolution ion mobility experiments were performed on a modified SYNAPT HDMS system fitted with a prototype cyclic IMS device and dual gain ADC. Data were processed in MassLynx, DriftScope and UNIFI software.
Preliminary Data or Plenary Speakers Abstract
SEC-native-IMS-MS was conducted to gain structural insights into the biotherapeutics trastuzumab and trastuzumab-emtansine. Trastuzumab showed a single charge series centered at approximately 6500 m/z, with excellent glycoform resolution. Ion mobility arrival time distributions (ATDs) were indicative of a single group of conformations, confirming that the folded structure of the antibody can be maintained using this standard-flow SEC-IMS-MS method. Under reducing conditions small amounts of native trastuzumab light chain and half-antibody were observed, made possible due to the ability of ion mobility to separate analytes from background ions. Ion mobility-mass spectra of non-deglycosylated trastuzumab-emtansine were complex, made up of different glycoforms and drug load variants, and were used to calculate the drug-to-antibody ratio. ATDs again indicated a single group of gas phase structures, and showed similarity to those of trastuzumab. Under reducing conditions native light chain and light chain with one or two conjugations were observed. These light chain species each displayed distinct ATDs, indicative of the differences in structure associated with the conjugation. In addition, for both biotherapeutics, elevated energies were used to cause collision-induced dissociation of the reduced antibody. This allowed observation of intact light chain and heavy chain product ions at low m/z, meaning masses of the whole antibody and both chains (with their corresponding conjugations) could be obtained in a single SEC-native-MS experiment. To investigate further the structures of the biotherapeutics, we conducted experiments on a cyclic IMS-enabled research platform. With this set-up it is possible to enhance ion mobility resolution by increasing the number passes through the cyclic IMS device. Trastuzumab ions, introduced by nanoESI, could be transmitted by at least three passes around the cyclical IMS device (3 m path length) with minimal ion losses, showing promise for high m/z species. Further investigations on the benefits of cyclic IMS for biotherapeutic analysis will be discussed.
Novel Aspect
Conducting online native sec-ion mobility-MS of biotherapeutics on a routine platform and nanoflow infusion on a cyclic IMS-enabled research platform.
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