Article 2024 · 0 atıf

Exploring the structural heterogeneity of IgG1s: A comprehensive analysis of glycosylated and deglycosylated forms using TIMS-TOF-MS technique

Ozturk, Serhat, Atakay, Mehmet, Avci, Izzet, Baltaci, Sema, Yaman, Mehmet Emrah, Seckin, Ali Ihsan, Salih, Bekir

Özet

Denaturation of IgG is a complex and multistep process that involves various structural changes in the protein as it loses its native conformation. IgG denaturation is crucial in therapeutic antibody development, manufacturing, and storage. Glycosylated IgG is generally more resistant to denaturing conditions due to the stabilizing effect of its glycans. In contrast, deglycosylated IgG is more vulnerable to structural disruption. In this study, we examined the intact level and large fragments of IgG glycoforms. Our investigation focused on changes in structural heterogeneity through glycosylation under denaturing conditions. Collision cross-sections (CCS) of intact level and large fragments of IgG1 molecules have been determined using reversed phase-high performance liquid chromatography-electrospray ionization-trapped ion mobility-time-of-flight mass spectrometry (RP-HPLC-ESITIMS-TOF-MS) technique. At the intact level, deglycosylated bevacizumab (IgG1B) and trastuzumab (IgG1T) could be differentiated at specific charge states and CCS ranges. The deglycosylated IgG1B molecule showed a broader CCS range than the IgG1T molecule. This result demonstrated that the deglycosylated IgG1T was altered from deglycosylated IgG1B in the gas phase under denaturing conditions. The lowering in the collision crosssection distribution (CCSD) values indicates a decrease in the structural heterogeneity of the intact IgG after deglycosylation. The ion mobility-mass spectrometry (IM-MS) analyses performed in this study showed that the glycans on the Fc region of IgG1s can trigger structural changes in the molecule under denaturing conditions. Depending on whether they are glycosylated or deglycosylated, various conformations of IgG1 molecules could be distinguished in RP-HPLC-TIMS-TOF-MS analysis.

Anahtar Kelimeler

Immunoglobulin G Denaturation Glycosylation Mass spectrometry Trapped ion mobility spectrometry MOBILITY-MASS-SPECTROMETRY MONOCLONAL-ANTIBODY N-GLYCOSYLATION AGGREGATION STABILITY DENATURATION EXCHANGE CONFORMATION FLUORESCENCE DYNAMICS

WoS Kategorileri

Chemistry, Analytical

Sürdürülebilir Kalkınma Amaçları

Sağlık ve Kaliteli Yaşam

Künye

Dergi: MICROCHEMICAL JOURNAL
Yıl: 2024
Cilt: 207
ISSN: 0026-265X
eISSN: 1095-9149
DOI: 10.1016/j.microc.2024.112099
WoS UID: WOS:001355957800001

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