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Successive remodeling of IgG glycans using a solid-phase enzymatic platform.

ABSTRACT: The success of glycoprotein-based drugs in various disease treatments has become widespread. Frequently, therapeutic glycoproteins exhibit a heterogeneous array of glycans that are intended to mimic human glycopatterns. While immunogenic responses to biologic drugs are uncommon, enabling exquisite control of glycosylation with minimized microheterogeneity would improve their safety, efficacy and bioavailability. Therefore, close attention has been drawn to the development of glycoengineering strategies to control the glycan structures. With the accumulation of knowledge about the glycan biosynthesis enzymes, enzymatic glycan remodeling provides a potential strategy to construct highly ordered glycans with improved efficiency and biocompatibility. In this study, we quantitatively evaluate more than 30 enzymes for glycoengineering immobilized immunoglobulin G, an impactful glycoprotein class in the pharmaceutical field. We demonstrate successive glycan remodeling in a solid-phase platform, which enabled IgG glycan harmonization into a series of complex-type N-glycoforms with high yield and efficiency while retaining native IgG binding affinity.

SUBMITTER: Hsu YP

PROVIDER: S-EPMC8990068 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Successive remodeling of IgG glycans using a solid-phase enzymatic platform.

Hsu Yen-Pang YP Verma Deeptak D Sun Shuwen S McGregor Caroline C Mangion Ian I Mann Benjamin F BF

Communications biology 20220407 1

The success of glycoprotein-based drugs in various disease treatments has become widespread. Frequently, therapeutic glycoproteins exhibit a heterogeneous array of glycans that are intended to mimic human glycopatterns. While immunogenic responses to biologic drugs are uncommon, enabling exquisite control of glycosylation with minimized microheterogeneity would improve their safety, efficacy and bioavailability. Therefore, close attention has been drawn to the development of glycoengineering str ...[more]

PMID: 35393560

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Project description:The sample preparation step is pivotal in glycoproteomic analysis. An effective approach in glycoprotein sample preparation involves enriching glycopeptides by solid-phase extraction (SPE) using polar stationary phases in hydrophilic interaction liquid chromatography (HILIC) mode. The aim of this work is to show how different experimental conditions influence the enrichment efficiency of glycopeptides from human immunoglobulin G (IgG) on an aminopropyl-modified SPE column. Different compositions of the elution solvent (acetonitrile, methanol, and isopropanol), along with varying concentrations of elution solvent acidifiers (formic and acetic acid), and different concentrations of acetonitrile for the conditioning and washing solvents (65%, 75%, and 85% acetonitrile) were tested to observe their effects on the glycopeptide enrichment process. Isopropanol proved less effective in enriching glycopeptides, while acetonitrile was the most efficient, with methanol in between. Higher formic acid concentrations in the elution solvent weakened the ionic interactions, particularly with sialylated glycopeptides. Substituting formic acid with acetic acid led to earlier elution of more glycopeptides. The acetonitrile concentration in conditioning and washing solutions played a key role; at 65% acetonitrile, glycopeptides were not retained on the SPE column and were detected in the flow-through fraction. Ultimately, it was proven that the enrichment method was applicable to human plasma samples, resulting in a significant decrease in the abundances of non-glycosylated peptides. To the best of our knowledge, this study represents the first systematic investigation into the impact of the mobile phase on glycopeptide enrichment using an aminopropyl-modified SPE column in HILIC mode. This study demonstrates the substantial impact of even minor variations in experimental conditions, which have not yet been considered in the literature, on SPE-HILIC glycopeptide enrichment. Consequently, meticulous optimization of these conditions is imperative to enhance the specificity and selectivity of glycoproteomic analysis, ensuring accurate and reliable quantification.

Dataset Information

Successive remodeling of IgG glycans using a solid-phase enzymatic platform.

Publications

Successive remodeling of IgG glycans using a solid-phase enzymatic platform.

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