It is now acknowledged that extracellular vesicles (EVs) are important effectors in a vast number of biological processes through intercellular transfer of biomolecules. Increasing research efforts in the EV field have yielded an appreciation for the potential role of glycans in EV function. Indeed, recent reports show that the presence of glycoconjugates is involved in EV biogenesis, in cellular recognition and in the efficient uptake of EVs by recipient cells. It is clear that a full understanding of EV biology will require researchers to focus also on EV glycosylation through glycomics approaches. This review outlines the major glycomics techniques that have been applied to EVs in the context of the recent findings. Beyond understanding the mechanisms by which EVs mediate their physiological functions, glycosylation also provides opportunities by which to engineer EVs for therapeutic and diagnostic purposes. Studies characterising the glycan composition of EVs have highlighted glycome changes in various disease states, thus indicating potential for EV glycans as diagnostic markers. Meanwhile, glycans have been targeted as molecular handles for affinity‐based isolation in both research and clinical contexts. An overview of current strategies to exploit EV glycosylation and a discussion of the implications of recent findings for the burgeoning EV industry follows the below review of glycomics and its application to EV biology.

Extracellular vesicles (EVs) are important mediators of cell-cell communication in a broad variety of physiological contexts. However, there is ambiguity around the fundamental mechanisms by which these effects are transduced, particularly in relation to their uptake by recipient cells. Multiple modes of cellular entry have been suggested and we have further explored the role of glycans as potential determinants of uptake, using EVs from the murine hepatic cell lines AML12 and MLP29 as independent yet comparable models. Lectin microarray technology was employed to define the surface glycosylation patterns of EVs. Glycosidases PNGase F and neuraminidase which cleave N-glycans and terminal sialic acids, respectively, were used to analyze the relevance of these modifications to EV surface glycans on the uptake of fluorescently labelled EVs by a panel of cells representing a variety of tissues. Flow cytometry revealed an increase in affinity for EVs modified by both glycosidase treatments. High-content screening exhibited a broader range of responses with different cell types preferring different vesicle glycosylation states. We also found differences in vesicle charge after treatment with glycosidases. We conclude that glycans are key players in the tuning of EV uptake, through charge-based effects, direct glycan recognition or both, supporting glycoengineering as a toolkit for therapy development.

Cell-secreted extracellular vesicles (EVs) have rapidly gained prominence as sources of biomarkers for non-invasive biopsies, owing to their ubiquity across human biofluids and physiological stability. There are many characterisation studies directed towards their protein, nucleic acid, lipid and glycan content, but more recently the metabolomic analysis of EV content has also gained traction. Several EV metabolite biomarker candidates have been identified across a range of diseases, including liver disease and cancers of the prostate and pancreas. Beyond clinical applications, metabolomics has also elucidated possible mechanisms of action underlying EV function, such as the arginase-mediated relaxation of pulmonary arteries or the delivery of nutrients to tumours by vesicles. However, whilst the value of EV metabolomics is clear, there are challenges inherent to working with these entities—particularly in relation to sample production and preparation. The biomolecular composition of EVs is known to change drastically depending on the isolation method used, and recent evidence has demonstrated that changes in cell culture systems impact upon the metabolome of the resulting EVs. This review aims to collect recent advances in the EV metabolomics field whilst also introducing researchers interested in this area to practical pitfalls in applying metabolomics to EV studies.

Eukaryotic cell‐free protein synthesis (CFPS) can accelerate expression and high‐throughput analysis of complex proteins with functionally relevant post‐translational modifications (PTMs). However, low yields and difficulties scaling such systems have prevented their widespread adoption in protein research and manufacturing.Here, we provide detailed demonstrations for the capabilities of a CFPS system derived from Nicotiana tabacum BY‐2 cell culture (BY‐2 lysate; BYL). BYL is able to express diverse, functional proteins at high yields in 48 h, complete with native disulfide bonds and N‐glycosylation. An optimized version of the technology is commercialized as ALiCE® and advances in scaling of BYL production methodologies now allow scaling of eukaryotic CFPS reactions. We show linear, lossless scale‐up of batch mode protein expression from 100 µL microtiter plates to 10 and 100 mL volumes in Erlenmeyer flasks, culminating in preliminary data from a litre‐scale reaction in a rocking‐type bioreactor. Together, scaling across a 20,000x range is achieved without impacting product yields.Production of multimeric virus‐like particles from the BYL cytosolic fraction were then shown, followed by functional expression of multiple classes of complex, difficult‐to‐express proteins using the native microsomes of the BYL CFPS. Specifically: a dimeric enzyme; a monoclonal antibody; the SARS‐CoV‐2 receptor‐binding domain; a human growth factor; and a G protein‐coupled receptor membrane protein. Functional binding and activity are demonstrated, together with in‐depth PTM characterization of purified proteins through disulfide bond and N‐glycan analysis.Taken together, BYL is a promising end‐to‐end R&D to manufacturing platform with the potential to significantly reduce the time‐to‐market for high value proteins and biologics.

De novo protein binders can now be generated far faster than they can be tested. Current validation routes — display selection, cell-free two-hybrid assays, or surface-based biophysics — require cell culture, colony picking, protein purification, target labeling or sensor immobilisation, take 2 to 8 weeks, and require additional methods for affinity and kinetics characterisation. Here we report an end-to-end, cell- and purification-free pipeline that goes from gene to binding affinity and kinetics in three days without genetically modified organisms, cell culture, purification, labelling or immobilisation. An automated, metric-driven loop of RFdiffusion, ProteinMPNN and AlphaFold2 initial-guess generates and filters designs, which are ranked by Boltz-2 ipSAE, assembled by Golden Gate cloning from linear DNA, expressed in a cell-free protein synthesis lysate as fluorescent protein fusions, and screened directly in the unpurified product by flow-induced dispersion analysis (FIDA). Against human growth hormone, 11 of 30 designs (37%) bound, and the four strongest had equilibrium dissociation constants of 0.93–9.98 nM with on- and off-rates resolved from the same cell-free material. Each sample run measures expression level, hydrodynamic radius, oligomeric and aggregation state alongside the binding readout, producing the standardised, per-design datasets that model training requires.