Toward Rapid Aspartic Acid Isomer Localization in Therapeutic Peptides Using Cyclic Ion Mobility Mass Spectrometry.

There is an increasing emphasis on the critical evaluation of interbatch purity and physical stability of therapeutic peptides. This is due to concerns over the impact that product- and process-related impurities may have on safety and efficacy of this class of drug. Aspartic acid isomerization to isoaspartic acid is a common isobaric impurity that can be very difficult to identify without first synthesizing isoAsp peptide standards for comparison by chromatography. As such, analytical tools that can determine if an Asp residue has isomerized, as well as the site of isomerization within the peptide sequence, are highly sought after. Ion mobility-mass spectrometry is a conformation-selective method that has developed rapidly in recent years particularly with the commercialization of traveling wave ion mobility instruments. This study employed a cyclic ion mobility (cIMS) mass spectrometry system to investigate the conformational characteristics of a therapeutic peptide and three synthetic isomeric forms, each with a single Asp residue isomerized to isoAsp. cIMS was able to not only show distinct conformational differences between each peptide but crucially, in conjunction with a simple workflow for comparing ion mobility data, it correctly located which Asp residue in each peptide had isomerized to isoAsp. This work highlights the value of cIMS as a potential screening tool in the analysis of therapeutic peptides prone to the formation of isoAsp impurities.

Galactose Oxidase Enables Modular Assembly of Conjugates from Native Antibodies with High Drug-to-Antibody Ratios.

The potential of antibody conjugates with high drug loading in anticancer therapy has recently been highlighted by the approval of Trastuzumab deruxtecan and Sacituzumab govitecan. These biopharmaceutical approaches have spurred interest in bioconjugation strategies with high and defined degrees of drug-to-antibody ratio (DAR), in particular on native antibodies. Here, a glycoengineering methodology was developed to generate antibody drug conjugates with DAR of up to eight, by combining highly selective enzymatic galactosylation and oxidation with biorthogonal tandem Knoevenagel-Michael addition chemistry. This four-step approach offers a selective route to conjugates from native antibodies with high drug loading, and thus illustrates how biocatalysis can be used for the generation of biopharmaceuticals using mild reaction conditions.

Utility of Ion-Mobility Spectrometry for Deducing Branching of Multiply Charged Glycans and Glycopeptides in a High-Throughput Positive ion LC-FLR-IMS-MS Workflow.

High-throughput glycan analysis has become an important part of biopharmaceutical production and quality control. However, it is still a significant challenge in the field of glycomics to easily deduce isomeric glycan structures, especially in a high-throughput manner. Ion mobility spectrometry (IMS) is an excellent tool for differentiating isomeric glycan structures. However, demonstrations of the utility of IMS in high-throughput workflows such as liquid chromatography-fluorescence-mass spectrometry (LC-FLR-MS) workflows have been limited with only a small amount of collision cross section (CCS) data available. In particular, IMS data of glycan fragments obtained in positive ion mode are limited in comparison to those obtained in negative ion mode despite positive ion mode being widely used for glycomics. Here, we describe IMS TWCCSN2 data obtained from a high-throughput LC-FLR-IMS-MS workflow in positive ion mode. We obtained IMS data from a selection of RapiFluor-MS (RFMS) labeled N-glycans and also glycopeptides. We describe how IMS is able to distinguish isomeric N-glycans and glycopeptides using both intact IMS and fragment-based IMS glycan sequencing experiments in positive ion mode, without significantly altering the high-throughput nature of the analysis. For the first time, we were able to successfully use IMS in positive ion mode to determine the branching of isomeric glycopeptides and RFMS labeled glycans. Further, we highlight that IMS glycan sequencing of fragments obtained from RFMS labeled glycans was similar to that of glycopeptides. Finally, we show that the IMS glycan sequencing approach can highlight shared structural features of nonisomeric glycans in a high-throughput LC-FLR-IMS-MS workflow.

Enzyme promiscuity of carbohydrate active enzymes and their applications in biocatalysis.

The application of biocatalysis for the synthesis of glycans and glycoconjugates is a well-established and successful strategy, both for small and large scale synthesis. Compared to chemical synthesis, is has the advantage of high selectivity, but biocatalysis had been largely limited to natural glycans both in terms of reactivity and substrates. This review describes recent advances in exploiting enzyme promiscuity to expand the range of substrates and reactions that carbohydrate active enzymes (CAZymes) can catalyse. The main focus is on formation and hydrolysis of glycosidic linkages, including sugar kinases, reactions that are central to glycobiotechnology. In addition, biocatalysts that generate sugar analogues and modify carbohydrates, such as oxidases, transaminases and acylases are reviewed. As carbohydrate active enzymes become more accessible and protein engineering strategies become faster, the application of biocatalysis in the generation of a wide range of glycoconjugates, beyond natural structures is expected to expand.

Biochemical characterisation of an α1,4 galactosyltransferase from Neisseria weaveri for the synthesis of α1,4-linked galactosides.

The human cell surface trisaccharide motifs globotriose and P1 antigen play key roles in infections by pathogenic bacteria, which makes them important synthetic targets as antibacterial agents. Enzymatic strategies to install the terminal α1,4-galactosidic linkage are very attractive but have only been demonstrated for a limited set of analogues. Herein, a new bacterial α1,4 galactosyltransferase from N. weaveri was cloned and produced recombinantly in E. coli BL21 (DE3) cells, followed by investigation of its substrate specificity. We demonstrate that the enzyme can tolerate galactosamine (GalN) and also 6-deoxygalactose and 6-deoxy-6-fluorogalactose as donors, and lactose and N-acetyllactosamine as acceptors, leading directly to analogues of Gb3 and P1 that are valuable chemical probes and showcase how biocatalysis can provide fast access to a number of unnatural carbohydrate analogues.

Exploiting the Disialyl Galactose Activity of α2,6-Sialyltransferase from Photobacterium damselae To Generate a Highly Sialylated Recombinant α-1-Antitrypsin.

Sialic acids are sugars present in many animal glycoproteins and are of particular interest in biopharmaceuticals, where a lack of sialylation can reduce bioactivity. Here, we describe how α-2,6-sialyltransferase from Photobacterium damselae can be used to markedly increase the level of sialylation of CHO-produced α-1-antitrypsin. Detailed analysis of the sialylation products showed that in addition to the expected α-2,6-sialylation of galactose, a second disialyl galactose motif Neu5Ac-α2,3(Neu5Ac-α2,6)Gal was produced, which, to our knowledge, had never been detected on a mammalian glycoprotein. We exploited this disialyl galactose activity of the P. damselae in a multienzyme reaction to produce a highly sialylated α-1-antitrypsin. The influence of this unique disialylation on the in vitro activity of α-1-antitrypsin was studied, and a toolkit of mass spectrometry methods for identifying this new disialyl galactose motif in complex mixtures was developed.

Enzymatic synthesis of N-acetyllactosamine from lactose enabled by recombinant ß1,4-galactosyltransferases.

Utilising a fast and sensitive screening method based on imidazolium-tagged probes, we report unprecedented reversible activity of bacterial ß1,4-galactosyltransferases to catalyse the transgalactosylation from lactose to N-acetylglucosamine to form N-acetyllactosamine in the presence of UDP. The process is demonstrated by the preparative scale synthesis of pNP-ß-LacNAc from lactose using ß1,4-galactosyltransferase NmLgtB-B as the only biocatalyst.

Applications of a highly α2,6-selective pseudosialidase.

Within human biology, combinations of regioisomeric motifs of α2,6- or α2,3-sialic acids linked to galactose are frequently observed attached to glycoconjugates. These include glycoproteins and glycolipids, with each linkage carrying distinct biological information and function. Microbial linkage-specific sialidases have become important tools for studying the role of these sialosides in complex biological settings, as well as being used as biocatalysts for glycoengineering. However, currently, there is no α2,6-specific sialidase available. This gap has been addressed herein by exploiting the ability of a Photobacterium sp. α2,6-sialyltransferase to catalyze trans-sialidation reversibly and in a highly linkage-specific manner, acting as a pseudosialidase in the presence of cytidine monophosphate. Selective, near quantitative removal of α2,6-linked sialic acids was achieved from a wide range of sialosides including small molecules conjugates, simple glycan, glycopeptide and finally complex glycoprotein including both linkages.

Characterisation of a Bacterial Galactokinase with High Activity and Broad Substrate Tolerance for Chemoenzymatic Synthesis of 6-Aminogalactose-1-Phosphate and Analogues.

Glycosyl phosphates are important intermediates in many metabolic pathways and are substrates for diverse carbohydrate-active enzymes. Thus, there is a need to develop libraries of structurally similar analogues that can be used as selective chemical probes in glycomics. Here, we explore chemoenzymatic cascades for the fast generation of glycosyl phosphate libraries without protecting-group strategies. The key enzyme is a new bacterial galactokinase (LgGalK) cloned from Leminorella grimontii, which was produced in Escherichia coli and shown to catalyse 1-phosphorylation of galactose. LgGalK displayed a broad substrate tolerance, being able to catalyse the 1-phosphorylation of a number of galactose analogues, including 3-deoxy-3-fluorogalactose and 4-deoxy-4-fluorogalactose, which were first reported to be substrates for wild-type galactokinase. LgGalK and galactose oxidase variant M1 were combined in a one-pot, two-step system to synthesise 6-oxogalactose-1-phosphate and 6-oxo-2-fluorogalactose-1-phosphate, which were subsequently used to produce a panel of 30 substituted 6-aminogalactose-1-phosphate derivatives by chemical reductive amination in a one-pot, three-step chemoenzymatic process.

Label-Free Discovery Array Platform for the Characterization of Glycan Binding Proteins and Glycoproteins.

The identification of carbohydrate-protein interactions is central to our understanding of the roles of cell-surface carbohydrates (the glycocalyx), fundamental for cell-recognition events. Therefore, there is a need for fast high-throughput biochemical tools to capture the complexity of these biological interactions. Here, we describe a rapid method for qualitative label-free detection of carbohydrate-protein interactions on arrays of simple synthetic glycans, more complex natural glycosaminoglycans (GAG), and lectins/carbohydrate binding proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The platform can unequivocally identify proteins that are captured from either purified or complex sample mixtures, including biofluids. Identification of proteins bound to the functionalized array is achieved by analyzing either the intact protein mass or, after on-chip proteolytic digestion, the peptide mass fingerprint and/or tandem mass spectrometry of selected peptides, which can yield highly diagnostic sequence information. The platform described here should be a valuable addition to the limited analytical toolbox that is currently available for glycomics.