Mass spectrometry for the identification and analysis of highly complex glycosylation of therapeutic or pathogenic proteins

ABSTRACT

Introduction

Protein glycosylation influences characteristics such as folding, stability, protein interactions, and solubility. Therefore, glycan moieties of therapeutic proteins and proteins that are likely associated with disease pathogenesis should be analyzed in-depth, including glycan heterogeneity and modification sites. Recent advances in analytical methods and instrumentation have enabled comprehensive characterization of highly complex glycosylated proteins.

Area covered

The following aspects should be considered when analyzing glycosylated proteins: sample preparation, chromatographic separation, mass spectrometry (MS) and fragmentation methods, and bioinformatics, such as software solutions for data analyses. Notably, analysis of glycoproteins with heavily sialylated glycans or multiple glycosylation sites requires special considerations. Here, we discuss recent methodological advances in MS that provide detailed characterization of heterogeneous glycoproteins.

Expert opinion

As characterization of complex glycosylated proteins is still analytically challenging, the function or pathophysiological significance of these proteins is not fully understood. To reproducibly produce desired forms of therapeutic glycoproteins or to fully elucidate disease-specific patterns of protein glycosylation, a highly reproducible and robust analytical platform(s) should be established. In addition to advances in MS instrumentation, optimization of analytical and bioinformatics methods and utilization of glycoprotein/glycopeptide standards is desirable. Ultimately, we envision that an automated high-throughput MS analysis will provide additional power to clinical studies and precision medicine.

KEYWORDS:

• N-glycosylation
• O-glycosylation
• immunoglobulin glycosylation
• Mucin 1 (MUC-1)
• virus glycoconjugates
• intravenous immunoglobulin (IVIG)
• Fc fusions protein therapeutics
• erythropoietin

Article highlights

• Glycans on therapeutic proteins or pathogenic proteins should be analyzed not only for glycoform profiles, but also for their site specificities.

• Glyco-engineering of therapeutic proteins leads to better clinical efficacy and less side effects.

• Glycosylation of therapeutic proteins should be monitored carefully, as glycans affect efficacy or toxicity.

• Advances in MS instrumentation and a series of analytical methods enable in-depth characterization of heavily glycosylated proteins, such as IgA.

• Development of high-throughput characterization of glycosylated proteins in clinical samples could be applicable for precision medicine.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Declaration of interest

The authors have no relevant affiliation or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. In the interest of full disclosure, we report that M. B. Renfrow and J. Novak are co-founders and co-owners of Reliant Glycosciences, LLC and co‑inventors on the US patent application 14/318,082 (assigned to UAB Research Foundation that distributes royalties to the inventors).

Additional information

Funding

The works described in this manuscript that were completed in our laboratories was supported by the JSPS KAKENHI grant numbers [16K09632, 19K08715, and 19K08691]; by a Grant-in-Aid for Practical Research Project for Renal Diseases, from the Japan Agency for Medical Research and Development; by Takeda Science Foundation; by Aichi Jinzou Foundation; and in part by NIH grants [DK078244, GM098539, DK105124, AI149431, and DK082753].