Defined Conjugation of Glycans to the Lysines of CRM₁₉₇ Guided by their Reactivity Mapping

Abstract

Systematic characterisation of the reactivity of the lysine moieties in CRM197 towards N-hydroxysuccinimide linkers bearing alkynes or azides is described. This involves two-step conjugation of various glycans to CRM197 by click chemistry in a well-defined manner. By semiquantitative LC-MS/MS analysis of proteolytic digests of the conjugates formed, the reactivity of lysine residues in the protein was mapped and ranked. Computational analysis of the solvent accessibility of each lysine residue (based on the CRM197 crystal structure) established a correlation between reactivity and surface exposure. By this approach, conjugation involving lysine residues (normally a random process) can be controlled. It enables the preparation of lysine-mediated glycoconjugates with improved batch-to-batch reproducibility, thereby producing neo-glycoconjugates with more-consistent biological activity.

Advances in Chemical Protein Modification

“…Thus, a careful control of reaction conditions allows traditional, robust, and operationally simple albeit a priori nonselective Lys modification protocols to be used with a level of regioselectivity as recently demonstrated by Adamo and co-workers. 21 In this study, the authors have developed reproducible batch-to-batch Lys alkylations of CRM 197 after mapping their reactivity profile using semiquantitative LC-MS/MS as well as computational analyses. Similarly, Weil and co-workers reported a site-and regioselective Lys modification protocol via kinetically controlled acylation by stepwise addition of substoichiometric amounts of an activated N-hydroxysuccinimide (NHS) ester under mild conditions.…”

The expanding role of mass spectrometry in the field of vaccine development

“…In one of the studies, researchers used LC‐MS to characterize glycosylated lysine residues in Menveo ® . In another study a LC‐MS method was used to determine the relative reactivity of lysine residues in CRM 197 to determine which of these amino acids were more susceptible to conjugation . LC‐MS was also used to quantify the Bexsero® vaccine which was the first vaccine developed by reverse vaccinology; a genome‐based approach to vaccine development .…”

Site‐Selective Functionalization of Flagellin by Steric Self‐Protection: A Strategy To Facilitate Flagellin as a Self‐Adjuvanting Carrier in Conjugate Vaccine

“…In contrast, the hypervariable D2 and D3 domains are arranged outside the flagellar filament, away from the binding interface of the FliC–TLR5 complex . Adamo and co‐workers recently found that the homodimer interface of CRM197 functioned as a noncovalent self‐protecting group . A study by Kolarich and co‐workers also illustrated that the local environment significantly influenced which lysine residues of CRM197 was modified .…”