Ion Mobility and Mass Spectrometry Measurements of the Humanized IgGk NIST Monoclonal Antibody

Iain D.G. Campuzano, Carlos Larriba, Dhanashri Bagal, Paul D. Schnier

Research output: Chapter in Book/Report/Conference proceedingConference contribution

25 Scopus citations

Abstract

Monoclonal antibodies are an important class of therapeutic agent that are in widespread use for the effective treatment of many human diseases. We have used a number of mass spectrometers (Synapt G2 and OrbiTrap Exactive Plus extended mass range [EMR]) and ion mobility instruments (Synapt G1 modified to an radio frequency [RF]-confining drift cell and a Synapt G2 travelling wave system) to characterise the humanised IgG1k NIST monoclonal antibody molecule under native-MS and buffer conditions. On both the Synapt G2 and the OrbiTrap instrumentation, charge state distributions are very similar, and all major glycoforms were resolved on all observable charge states; however, only on the OrbiTrap instrument are the glycoforms fully resolved to baseline. Ion mobility measurements were made in both helium and nitrogen drift gases on an RF-confining drift cell device with derived collision cross-section values for charge states +21 to +26 range from 6696 2 to 6892 2 in helium and 7223 2 to 7403 2 in nitrogen, respectively. This small but gradual increase in collision cross section with increasing charge can be attributed to a combination of increasing ion-induced dipole interaction between the charged protein and the neutral drift gas and the enlargement of the protein due to the repulsion of the surface charges. The higher charge states (+27 to +29) show a significant amount of gas-phase unfolding, evident by increased collision cross-section values and bimodal ion mobility arrival times. Collision cross sections measured on a travelling wave device (in nitrogen) were consistent with the mobility measurements made in nitrogen on the RF-confining drift cell device. Molecular dynamic simulations on the theoretically generated NIST monoclonal antibody (NISTmAb) coordinate structure indicate that the gas-phase structure undergoes a significant (up to 40%) amount of compaction. Theoretical collision cross-section calculations on the optimised molecular dynamic-derived NISTmAb structure are consistent with both helium and nitrogen instrument-derived collision cross-section values. Finally, collision-induced unfolding experiments also were performed on the +26 charge of the NISTmAb and could potentially be used as a means of providing structural information in addition to a single, and potentially limiting, gas phase-derived collision cross-section measurement.

Original languageEnglish (US)
Title of host publicationState-of-the-Art and Emerging Technologies for Therapeutic Monoclonal Antibody Characterization
Subtitle of host publicationVolume 3. Defining the Next Generation of Analytical and Biophysical Techniques
EditorsJohn E. Schiel, Oleg V. Borisov, Darryl L. Davis
PublisherAmerican Chemical Society
Pages75-112
Number of pages38
ISBN (Electronic)9780841230316
DOIs
StatePublished - Jan 1 2015

Publication series

NameACS Symposium Series
Volume1202
ISSN (Print)0097-6156
ISSN (Electronic)1947-5918

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