Unit mass baseline resolution for an intact 148 kDa therapeutic monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry

Abstract

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides the highest mass resolving power and mass measurement accuracy for unambiguous identification of biomolecules. Previously, the highest-mass protein for which FTICR unit mass resolution had been obtained was 115 kDa at 7 T. Here, we present baseline resolution for an intact 147.7 kDa monoclonal antibody (mAb), by prior dissociation of noncovalent adducts, optimization of detected total ion number, and optimization of ICR cell parameters to minimize space charge shifts, peak coalescence, and destructive ion cloud Coulombic interactions. The resultant long ICR transient lifetime (as high as 20 s) results in magnitude-mode mass resolving power of ~420,000 at m/z 2,593 for the 57+ charge state (the highest mass for which baseline unit mass resolution has been achieved), auguring for future characterization of even larger intact proteins and protein complexes by FTICR MS. We also demonstrate up to 80% higher resolving power by phase correction to yield an absorption-mode mass spectrum.

Figure 1

Schematic representation of the structure and glycoforms for recombinant monoclonal antibody, IgG1k.

Figure 2

Broadband positive ESI 9.4 T FT-ICR MS mass spectrum for an IgG1k antibody protein. The characteristic charge state distribution envelope from 47+ to 67+ (m/z 2,200 – 3,200) is achieved by front-end skimmer dissociation of non-covalent adducts.

Figure 3

Top: Positive ESI 9.4 T magnitude-mode FT-ICR mass spectrum for quadrupole-selected IgG1k showing 57+ charge state molecular ions along with various adducts, based on an 11.6 s transient with 5 isotopic beats (inset). Bottom: Mass scale-expanded segment, demonstrating unit mass baseline resolution of the isotopic distribution at ~290,000 average resolving power.

Figure 4

Calculated and experimental isotopic distributions for multiply-charged (57+) IgG1k molecular ions. The inset table shows the chi-square values obtained by shifting the theoretical distribution by ~1/z (i.e., 1 Da) increments to the left and right relative to the experimental distribution to locate the best fit with least error.^, The calculated mass is for the unadducted antibody with two G0F glycans (see Figure 1)

Figure 5

ESI 9.4 T FT-ICR mass spectra for the IgG1k antibody. Top: magnitude mode; Bottom: absorption mode. Phase correction of the magnitude-mode mass spectrum results in an absorption-mode spectrum with ~1.8 times higher resolving power.