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.

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.

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.