Posters

Field-asymmetric ion mobility spectrometry to improve the separation and identification of released N-glycans

Carbohydrates are among the most abundant and diverse compounds found in nature. They are involved in many biological processes and serve as markers for various diseases. As glycosylation is understood as a non-template driven process, the resulting glycans span a wide diversity of structures with inherent variations in composition, conformation, size, and polarity. Added to low and uneven ionization efficiencies, these aspects make their analysis particularly challenging.

Morsa, D., Szabo, Z., Boeser, C.L.

71^st ASMS Conference on Mass Spectrometry and Allied Topics (2023), Houston, TX

Characterization of glycosylation in biopharmaceuticals using SimGlycan^® software for LC-MS/MS analysis of released glycans

Biopharmaceuticals renowned for the treatment of cancer and other diseases, are the recombinant glycoproteins produced using biological expression systems. Micro and macro-heterogeneities in glycosylation are strongly associated with stability, safety, and efficacy of these biopharmaceuticals. Thus, adequate characterization of critical glycosylation features of therapeutic glycoproteins is indispensable. However, the inherent complexities of protein glycosylation pose daunting analytical challenges.

Puranik, A., Goswami, R., Sutar, P., Tupe, D., Rasam, P., Dandekar, P., Jain, R.,

8^th International Bioprocessing India (BPI) Conference 2022, Pune, India

Creating LC-MS Template for Rapid and Accurate Identification of Cetuximab N-Glycans using UHPLC Coupled with Orbitrap-Based MS using SimGlycan Software

Cetuximab, a chimeric IgG1 monoclonal antibody (mAb), primarily targets epidermal growth factor receptor and thus are used in treatment of head and neck cancer, and colorectal cancer[1]. Understanding the structural complexity of cetuximab is vital for expanding the therapeutic application of this antibody. Liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) technique has been most commonly used for glycan characterization [2]. LC-MS methods generate large volumes of data and therefore require the use of sophisticated bioinformatics tools. SimGlycan software [3,4] facilitates LC-MS data processing, and automated identification of glycan compositional structures by annotating in silico fragment ions on peaks of the MS2 spectra.

Identification of hIgG N-Glycans Labeled with Procainamide via Reductive Amination using UHPLC Coupled with an Orbitrap-based Mass Spectrometer

Immunoglobulin G (IgG) represents approximately 75% of serum antibodies in humans. Clinically, measured IgG antibody levels are generally considered to be indicative of an individual's immune status to particular pathogens. Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. IgG molecules bear oligosaccharides that play an essential role in Fc effector functions including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) [1].

Automated Identification and Quantitation of 2-AA Derivatized N-Glycans from Infliximab Using UHPLC-Orbitrap-MS Analysis with SimGlycan Software

Infliximab is a monoclonal IgG1 antibody designed to target tumor necrosis factor-alpha, a key inflammatory cytokine[1]. Glycosylation of Infliximab during recombinant synthesis must be fully characterized for therapeutic development. Liquid chromatography (LC)-mass spectrometry (MS) with tandem MS (MS/MS) based methods enables identification of monosaccharide compositions and linkage orientation of the glycans [2-4].

Meitei, N.S., Gupta, H., Apte, A., Widdowson, P., Millán, S., Carillo, S., Bones. J.,

67^th ASMS Conference on Mass Spectrometry and Allied Topics (2019),
Atlanta, GA

Automated Evaluation of Glycosylation Profile of Glycoengineered Monoclonal Antibodies using LC-MS Methods with SimGlycan

Monoclonal antibodies (mAbs) are being used increasingly for treating several diseases such as cancer, autoimmune diseases and other chronic diseases [1]. Most of the therapeutic mAbs are produced in yeast or mammalian cell lines that yield mixtures of Fc glycoforms, which are similar to human glycoforms but not completely identical. Glycoengineering is a promising technique to control the glycosylation profiles so as to improve safety, clinical efficacy, and effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) [2-3].

Meitei, N.S., Gupta, H.,

Applied Pharmaceutical Analysis (2019), Organized by The Boston Society, Goa, India

SimGlycan: Facilitating Automated Evaluation of Glycoengineered Therapeutic Glycoproteins using MS Methods

Monoclonal antibody (mAb) based therapeutics are the fastest growing class of human pharmaceuticals [1]. Variation in IgG Fc glycoforms affects the safety, clinical efficacy, and effector functions of therapeutic antibodies including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) e.g., the terminal galactose enhances CDC activity, but fucose significantly decreases ADCC [2].

Meitei, N.S., Goswami, R.

SFG 2018 Annual Meeting, New Orleans, Louisiana

Structural Identification, and Quantitation of Glycans Labeled with Dual Modifications Strategies from Complex Mixtures using LC-MS Workflows with SimGlycan^® Software

Glycans play a critical role in the therapeutic efficacy and toxicity of monoclonal antibody proteins. Recently developed integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), enables quantitative comparison between neutral and sialylated glycans simultaneously using mass spectrometry methods [1].

Meitei, N.S., Li, H., Saha, S., Goswami, R., Apte, A., Lee, R.S.

66^th ASMS Conference on Mass Spectrometry and Allied Topics (2018),
San Diego, CA

Automated Identification and Quantitation of Stable Isotope Labeled Released N-glycans by LC-MS with SimGlycan^® Software

Glycans play a critical quality attribute for the therapeutic monoclonal antibodies, where terminal sugars of the Fc glycans are responsible for determining their efficacy and safety. One of the emerging trends of glycomics research include quantitative and qualitative analysis of glycans labeled with tandem mass tags or isotope labels.

Automated Identification and Subsequent Relative Quantitation of Glycans Using Stable Isotope Labels by MS with SimGlycan^® Software

Recent trends in glycomics research show rapid growth of innovation related to accurate mass spectrometry based quantitative analysis of glycans. Stable isotope labels facilitate accurate identification and subsequent quantification of glycans using LC-MS or ESI-MS workflows.

Meitei, N.S.

SFG 2017 Annual Meeting, Portland, Oregon

Automated and Rapid Characterization of Low-Molecular-Weight Heparin Oligosaccharides Using TripleTOF 5600 Mass Spectrometer with SimGlycan Software

Low molecular weight heparins (LWMH) - Enoxaparin, Parnaparin, Certoparin, Deltaparin, Tinzaparin, etc. - have been reported to be the key drugs and are as safe and effective as Unfractionated Heparin (UFH) [1,4] which further necessitates in-depth study of new regulatory drugs. Despite its medical and biological importance, LWMHs are relatively uncharacterized in terms of its chemical structures due to its structural complexity.

SimGlycan®: Informatics Support for Automated Characterization of Low-Molecular-Weight Heparin Oligosaccharides Using MS2 Data from TripleTOF® 5600 Mass Spectrometer

Recent reports suggested that patent protection worth of $80 billion for several biosimilars, including Low molecular weight heparins (LWMH) - Enoxaparin, Parnaparin, Certoparin, Deltaparin, Tinzaparin, etc. - has been expired and abbreviated new drug applications have been approved by different regulatory bodies, namely, the Food and Drug Administration (FDA), USA; Central Drugs Standard Control Organization (CDSCO), India, etc.

Speeding up the High Throughput Searches for Glycan Analysis

A typical LC-MS glycomic data processing includes peak detection, deconvolution of peaks of co-eluting isomers, molecular feature extraction, and selection of correct precursor m/z values from the isotope cluster for MS/MS data analysis.

Development of Bioinformatics support for Quantitative Glycomics using Tandem Mass Tags

AminoxyTMT reagents enable relative quantitation of glycans in a MS/MS spectrum. However, there are no bioinformatics tools to automate the qualitative/ quantitative analysis.

Profiling the Distribution of N-Glycosylation in Therapeutic Antibodies using the QTRAP^® 6500 System

Immunoglobulin G molecules have become attractive as targeted therapeutic proteins, due to their high specificity and long circulation time. Glycosylation patterns determine the stability and bio-disposition of these recombinant protein drugs in vivo, as well as the efficacy, folding, binding affinity, specificity and pharmacokinetic properties.

SimGlycan: Informatics tool for creating glycan templates to enable high throughput glycan identification from Liquid Chromatogram - tandem mass spectrometry data

Protein glycosylation is one of the well-known post-translational modifications, and in biological processes in living organisms it plays an important role. Monitoring changes in the amount and structure of sugar chains makes it possible to monitor sugar chain modification of proteins and various diseases.

Development of Bioinformatics Support for High Throughput Isomeric Separation and the Structural Identification of Glycans by LC-MS

The separation of glycans by chromatography prior to MS analysis can reduce sample complexity, minimize ion suppression, and increase dynamic range and separation of structural isomers.

Informatics Support for Isomeric Separation and the Structural Identification of Labeled N-Glycans from Proteins

Develop a software to facilitate LC-MS and MS/MS glycan data that have been acquired for separation and identification of various complex N-linked glycans from proteins using a novel high resolution mixed-mode column and an Orbitrap Fusion Tribrid mass spectrometer.

Automating Mass Spectrometry-Based Quantitative Glycomics using Tandem Mass Tag (TMT) Reagents with SimGlycan

One of the emerging trends in glycomics research is the innovation related to accurate MS based quantitative analysis of glycans1-4. Recently, we have introduced aminoxyTMT Reagents, which enable efficient relative quantitation of carbohydrates, improved labeled-glycan ionization efficiency and increased analytical throughput.

Automated Glycan Structural Isomer Differentiation Using Bioinformatics Tool

Mass spectrometry (MS) has emerged as a powerful tool for the structural elucidation of glycans. The use of permethylation in combination with multistage fragmentation (MSⁿ) is a critical step in glycan structural characterization.

Integration of SimGlycan into Waters Glycan/Glycopeptide Analysis Workflow

Translating New Technologies in Improved Workflows for Biotherapeutic Analysis.