Monitoring antigenic protein integrity during glycoconjugate vaccine synthesis using capillary electrophoresis-mass spectrometry

Abstract

A capillary electrophoresis-mass spectrometry (CE-MS) method was developed for the characterization and integrity assessment of the Mycobacterium tuberculosis (MTB) antigens TB10.4 and Ag85B and their chemically produced glycoconjugates, which are glycovaccine candidates against tuberculosis (TB). In order to prevent protein adsorption to the inner capillary wall and to achieve efficient separation of the antigen proteoforms, a polyionic multilayer coating of polybrene-dextran sulfate-polybrene (PB-DS-PB) was used in combination with 1.5 M acetic acid as background electrolyte (BGE). Coupling of CE to high-resolution time-of-flight MS was achieved by a coaxial interface employing a sheath liquid of isopropanol-water (50:50, v/v) containing 0.1 % formic acid. The MTB antigens were exposed to experimental conditions used for chemical glycosylation (but no activated saccharide was added) in order to investigate their stability during glycovaccine production. CE-MS analysis revealed the presence of several closely related degradation products, including truncated, oxidized and conformational variants, which were assigned by accurate mass. Analysis of synthesized mannose conjugates of TB10.4 and Ag85B allowed the determination of the glycoform composition of the neo-glycoproteins next to the characterization of degradation products which were shown to be partly glycoconjugated. Moreover, the selectivity of CE-MS allowed specific detection of deamidated species (protein mass change of 1.0 Da only), indicating that chemical glycosylation increased susceptibility to deamidation. Overall, the results show that CE-MS represents a useful analytical tool for the detailed characterization and optimization of neo-glycoconjugate products. Graphical AbstractFlowchart illustrating Mycobacterium tuberculosis (MTB) antigen glycosylation, glycoconjugate variant and degradation product separation by capillary electrophoresis (CE) and their characterization by intact mass spectrometry (MS) Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9723-5) contains supplementary material, which is available to authorized users.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…Tengattini et al. used CZE‐MS for the intact characterization of synthetically produced glycoconjugate vaccines . The influence of the reaction conditions on the stability of the carrier proteins was evaluated.…”

Separation methods hyphenated to mass spectrometry for the characterization of the protein glycosylation at the intact level

“…Another cationic polymer containing an amine (LA 113) was also reported for capillary coating and the analysis of VGEF 165 isoforms [63]. A multilayer coating based on PB-dextran sulfate (DS)-PB was used to improve EOF reproducibility and coating stability [57,66].…”

“…The generated intensity and therefore the Joule effect is also a criterion and some limit values are recommended with some CE-MS interfaces. Other conventional CZE parameters such as capillary length, voltage, and/or temperature must also be optimized in parallel with the BGE composition, as was done for example for the analysis of neoglycoproteins [66].…”

Recent advances in microscale separation

“…Proteins, e.g., enzymes, hormones, monoclonal antibodies, antigens, recombinant vaccines, and glycosylated proteins, have emerged as important therapeutics for early diagnosis and the treatment of various diseases . Microscale separation technique (e.g., CE, CEC) coupled with high sensitive detection (e.g., MS) has proven to be a powerful analytical tool for the separation and characterization of intact proteins .…”