Simplified identification of disulfide, trisulfide, and thioether pairs with 213 nm UVPD

Abstract

Disulfide heterogeneity and other non-native crosslinks introduced during therapeutic antibody production and storage could have considerable negative effects on clinical efficacy, but tracking these modifications remains challenging. Analysis must also be carried out cautiously to avoid introduction of disulfide scrambling or reduction, necessitating the use of low pH digestion with less specific proteases. Herein we demonstrate that 213 nm ultraviolet photodissociation streamlines disulfide elucidation through bond-selective dissociation of sulfur-sulfur and carbon-sulfur bonds in combination with less specific backbone dissociation. Importantly, both types of fragmentation can be initiated in a single MS/MS activation stage. In addition to disulfide mapping, it is also shown that thioethers and trisulfides can be identified by characteristic fragmentation patterns. The photochemistry resulting from 213 nm excitation facilitates a simplified, two-tiered data processing approach that allows observation of all native disulfide bonds, scrambled disulfide bonds, and non-native sulfur-based linkages in a pepsin digest of Rituximab. Native disulfides represented the majority of bonds according to ion count, but the highly solvent-exposed heavy/light interchain disulfides were found to be most prone to modification. Production and storage methods that facilitate non-native links are discussed. Due to the importance of heavy and light chain connectivity for antibody structure and function, this region likely requires particular attention in terms of its influence on maintaining structural fidelity.

Figure 1.

Examples of 213 nm UVPD of disulfide bound peptide pairs extracted from a pepsin digest of Rituximab. a) MS² of a disulfide link between Cys133 and Cys193 with inset showing the characteristic disulfide triplet pattern. In addition to direct observation of disulfide pairs, sequence information from both chains is also seen. b) UVPD of a disulfide between Cys265 and Cys325 which produces fragments ^SP1/^SP2 and abundant backbone fragmentation. Peptides and fragments are color-coded, i.e. a₄ is a normal a₄ fragment from P1, whereas a₁₀^-S-S-P₁ corresponds to the a₁₀ fragment from ^SP2 with the full sequence of ^SP1 still attached.

Figure 2.

Examples of alternative crosslink fragmentation following UVPD activation. MS/MS of a) thioether and b) trisulfide

Figure 3.

Sequence and disulfide coverage for the peptic digestion of Rituximab. LCMS-MS identified regions of sequence and crosslinks are labeled by red text or lines, respectively. The identified hinge region at HC230-HC230 and HC233–233 disulfides are marked by red asterisks

Figure 4.

Extent of disulfide heterogeneity as approximated by EIC intensities.

Figure 5.

Crystal structure of a full IgG1 mAb. Heavy chain portions are illustrated in green while the light chains are blue. Zoomed inset of the hinge region shows both the heavy-light interchain disulfide links HC224-HC213 and heavy-heavy interchain links HC230-HC230 and HC233-HC233. These links are numbered as they would appear in Rituximab for illustrative purposes.

Scheme 1.

Possible complementary fragmentation pairs for a) disulfides b) thioethers and c) trisulfides. Note that depending on the crosslink type, heterolytic and homolytic fragmentation of the crosslink can occur. Simplified notations for each precursor ion and fragment are outlined by red boxes.