Site-specific glycan engineering (SSGE) refers to the precise and deliberate installation of glycan structures to a specific position in a protein. In contrast to global glycan engineering, which seeks to shift the overall glycoform landscape of a protein, SSGE allows each position on the protein to be customized and optimized independently of the others. SSGE can be accomplished through a number of routes, including genetic, chemoenzymatic, and bioorthogonal methods. Site-specific glycoengineering is anticipated to be a key enabling technology for a variety of next-generation biopharmaceuticals. For antibody-drug conjugates (ADCs), controlled drug conjugation must be site-specific in order to obtain homogeneous products with high DARs. For bispecific antibodies (bsAbs), site-specific glycan addition can be essential to prevent mispairing of monomers with high site-specific occupancy. Similarly, many synthetic vaccines have epitopes that need to be controlled at the single-molecule level in order to display the antigen in a desired form. Mass spectrometry (MS) glycoproteomics can be used to validate site-specific occupancy of engineered glycans and therefore identify low-abundance PTMs such as sulfation and O-acetylation added at specific sites on Ig glycoproteins as well as micro-heterogeneity these positions may display which can't be determined from bulk glycan analysis approaches. As MS-based site-resolved glycan analysis is anticipated to be required by regulatory agencies for new biologics, SSGE is expected to go from academic novelty to expected practice for the manufacture of glycoprotein therapeutics whose glycans can be rationally engineered for the therapeutic context, patient population, and mode of action of choice.
Site-specific glycan engineering is a related discipline that has evolved from the understanding that naturally occurring glycosylation will probabilistically glycosylate at all available sequons, resulting in a glycoform mixture that obfuscates structure–activity relationships and makes it challenging to achieve the same glycan profile consistently during manufacturing. This becomes particularly relevant for engineering complex modalities, such as bispecific antibodies, where a discrepancy in glycosylation between two heavy chains can lead to aggregation or altered PK. Site-specific glycan engineering approaches the problem from the premise that glycosylation should be a programmable feature, rather than an emergent one and can be achieved in 3 general ways: 1) Genetic introduction of consensus N-linked sequons at the desired site, 2) Chemoenzymatic remodeling after production, which often involves trimming glycans at undesired sites and re-building a defined glycan at the desired site or 3) Bioorthogonal chemical conjugation approaches utilizing chemically synthesized glycans with functional handles for conjugation. Regardless of the approach, robust analytical characterization is required to ensure site-specific occupancy is not confused with partially occupied or empty sites. This usually requires high-resolution MS with fragment ion analysis to unambiguously assign the glycan location. This can be important for antibody-drug conjugates, where non-site-specific glycosylation can result in heterogenous drug-to-antibody ratios outside of the therapeutic window. Recent work has shown that affinity-guided conjugation strategies can take advantage of site-specific glycan remodeling at evolutionarily conserved regions of the Fc to generate homogeneous conjugation sites in unmodified antibodies, without the need for substantial protein engineering. As complex protein modalities continue to increase in size and architectural complexity, site-specific glycan engineering will be the only route to orthogonalize multiple functional requirements within one molecule.
Fig. 1 Experimental design for expanding the N-glycoproteomic analysis of IgA by applying strategies for the identification of sulfated and other rare N-glycopeptides.1,5
Site-specific glycan engineering involves the incorporation of glycans in defined positions of proteins in a stoichiometric and site-selective manner, as a contrast to natural glycosylation that is stochastic and sub-stoichiometric. The three primary strategies to achieve site-specific glycosylation are genetic incorporation of N-linked glycosylation sequons (Asn-X-Ser/Thr) into target loops in a defined manner, chemoenzymatic remodeling of proteins in which heterogeneous glycans are removed to the core by endoglycosidases and then rebuilt with defined glycans using glycosyltransferases and bioorthogonal ligation of synthetic glycans, typically functionalized with an azide, alkyne, or another chemical handle for click chemistry, to genetically encoded protein handles like non-canonical amino acids or sortase binding motifs. While similar in name, site-specific glycan engineering is distinguished from glycoform engineering by the fact that it is non-stochastic and each protein molecule is glycosylated at defined sites in a stoichiometric manner, as opposed to altering the glycan profiles of a population of proteins. Site-specific glycan structures can be verified by advanced mass spectrometric glycoproteomics techniques which can provide site-specific glycan occupancy maps that can identify fully glycosylated, partially glycosylated and aglycosylated forms of glycopeptides with rare glycan modifications at specific positions (such as sulfated glycans, O-acetylated glycans, etc.) missed by bulk glycan analysis methods. Site-specific O-glycosylation is also possible, but requires more empirical determination in the absence of a strict sequon. Site-specific glycan engineering, in concept, treats glycans as a synthetic module whose function and activity is determined by its position on the protein of interest, allowing for the construction of therapeutic proteins that are custom built with glycans in specific positions on the protein tailored to the clinical need.
Site-specific glycosylation is important since the site of a glycan, as well as its structure, can determine the properties of a protein therapeutic. Misplaced glycans can sterically inhibit active sites, induce immunogenicity, or provide aggregation surfaces. For monoclonal antibodies, the site-specific placement of a core-fucose lacking glycan in the Fc region of the antibody can augment binding to Fc-γRIIIa and the efficacy of ADCC while maintaining antigen binding by the Fab. This spatially-segregated modification is not possible when using a heterogeneous mixture of glycoforms. Site-specific glycans can be used to control the stoichiometry of drug conjugation to antibody-drug conjugates. In this context, each site-specific glycan on the antibody can be used as a point of conjugation to add a defined number of drugs. Since stochastic glycosylation cannot be controlled this way, unwanted subpotent (sub-druged) or sub-cytotoxic (multi-druged) antibodies cannot be produced. Site-specific glycosylation can also be used to mask immunogenic epitopes, by placing a large oligosaccharide over exposed patches that may be different than native human glycoforms and therefore not recognized by the antibody. In bispecific antibody design, site-specific asymmetric glycosylation at the Fc interface can prevent heavy-chain mispairing during production, which is not possible with random, or stochastic, glycosylation. Site-specificity also provides mechanistic insight into glycan function by placing sugars on specific loops of the protein to identify which locations modulate receptor binding versus protease resistance. Site-resolved glycan information is also becoming a requirement for regulatory agencies where occupancy at each sequon is being treated as a unique critical quality attribute (CQA) for approval. As proteins with more functional domains become therapeutic proteins, site-specific glycosylation provides the only orthogonal strategy to tune these activities. For example, independent tuning of half-life and effector function is now possible.
Site-specific glycan modifications can be enzymatic or chemical in nature and allow for a defined carbohydrate moiety to be introduced at a specific position of a protein. In contrast to random glycosylation of proteins by the cell, site-specificity allows defined glycosylation patterns at any position. This is a desired improvement over traditional glycoengineering methods, which do not offer control over the subset of protein within a population to which a glycan will be added, as it allows for more easily characterized structure-function relationships, in addition to clearer regulatory requirements. Glycoengineering methods can be broadly divided into enzymatic methods and chemical methods. Enzymatic methods rely on the substrate specificity and reactivity of an enzyme, while chemical methods often utilize bioorthogonal chemistry, which can have an increased substrate scope and introduce unnatural chemical functionality. Enzymatic approaches make use of ligases (such as sortase, butelase, or variants such as peptiligase) that can recognize short peptide sequences and catalyze the formation of a peptide bond at the target site, typically allowing for the ligation of an engineered glycopeptide to the terminus of a protein with limited off-target reactivity. Chemical methods typically make use of bioorthogonal reactions (such as azide-alkyne cycloadditions, oxime ligations, or native chemical ligation) to install glycans onto genetically encoded handles, including non-canonical amino acids and cysteine residues introduced by site-directed mutagenesis. Many chemoenzymatic workflows have now been developed that use these strategies to convert recombinantly produced aglycosylated proteins to homogeneously glycosylated proteins with complex human-like N-glycans, a process that does not have the regulatory and immunogenicity limitations of mammalian cell culture and can be performed without loss of potency.
Chemical methods for controlling the position of glycans have been developed but often lack the desired positional specificity. By contrast, enzymes have evolved with high chemoselectivity and enzymatic methods for site-specific glycan engineering, such as those based on peptidoligases, have high positional selectivity. Peptidoligases catalyze peptide bond formation between a protein and a synthetic glycopeptide ester, affording a site-selectively attached N-acetylglucosamine (GlcNAc) residue for further elaboration by transglycosylation. Typically the protein of interest is expressed in a non-glycosylating host such as E. coli to yield a homogeneous, unmodified starting material. A peptidoligase enzyme ligates a synthetic glycopeptide with a GlcNAc tag to the N-terminus of the protein. The GlcNAc-tagged protein can then be acted upon by ENGase mutants that are specifically designed to only catalyze transglycosylation in order to transfer the sugar oxazoline onto the GlcNAc primer site. This process is orthogonal to high-yielding bacterial protein expression, allowing site-specific glycosylation of proteins without the need for mammalian cell culture. This can be used to glycosylate therapeutic proteins on demand, including cytokines and hormones, for example. The peptidoligase ligation reaction is often fast and high yielding, and sequence constraints on the target protein are minimal. Glycosylation reactions with the ENGases are often also fast and high yielding with conversions that are nearly quantitative. The specificity of the ENGase mutants used in the transfer reaction determine what glycan structures are transferred. For example, one can use a mutant that accepts biantennary sialoglycans or alternatively a mutant that will accept high mannose and hybrid glycans. A limitation in using peptidoligase ligation is the need to scale up the reaction, which can be challenging, due to cost and stability of the peptidoligase under Process conditions. Also the need for a synthetic glycopeptide donor requires chemical synthesis.
Chemical methods offer access to unnatural functions and linkages that are not supported by enzyme machinery, and are not limited to natural monosaccharide building blocks. The general strategy is to use bioorthogonal chemistry, that is, the introduction of an unnatural amino acid with an azide, alkyne, or ketone handle using amber codon suppression, which can then be used as a selective reactive handle to covalently link a synthetic glycan with a complementary tag. Azide-alkyne cycloaddition reactions are well established and can quantitatively ligate complex glycans to protein expressed in bacterial or mammalian cells with no cross-reactivity with canonical amino acids. Oxime ligation is another orthogonal strategy in which a ketone handle on the protein and a reactive aminooxy-glycan are combined in mildly acidic conditions to selectively form a stable oxime bond. This reaction is chemoselective, rapid, and effective at low concentrations, and has been used to site-specifically modify purified therapeutics. Native chemical ligation (NCL) has also been applied to site-specific glycosylation, by expressing the protein of interest with a C-terminal thioester that can react with a N-terminal cysteine on a synthetic glycopeptide, enabling direct installation of pre-synthesized glycans with defined linkages. Synthetic chemical strategies also allow more tolerance for synthetic modifications, allowing fluorinated sialic acids, click-functionalized GalNAc derivatives, and PEGylated glycans to be introduced to study structure-function relationships or for improved pharmacokinetic properties. An added advantage to chemical strategies is that they are applicable to proteins expressed in any host system, including bacterial cells which do not natively have any glycosylation and can thereby avoid immunogenic glycans as well as complex and expensive protein purification strategies to remove those glycans. On the downside, many chemical methods require protection of reactive functional groups, which adds additional synthetic steps, and reaction conditions, while mild, can in some cases cause protein denaturation or aggregation. The need for synthetic glycan donors can also add costs and regulatory overhead, as these must be manufactured GMP-compliant and fully characterized as starting materials. New methodologies such as aqueous-compatible click catalysts and site-selective cysteine alkylation under carefully buffered pH conditions are making it possible to modify proteins without prior protection in a single step.
Site-specific glycan engineering is a rapidly developing approach in modern biopharmaceuticals. In these applications, site-specificity is used to fine-tune the efficacy and pharmacokinetics of therapeutic glycoproteins beyond the capability of traditional bulk glycoengineering. For instance, in certain cases, site-specificity can be used to design glycoproteins with a specific glycan and specific glycan architecture at specific locations in 3D space on a protein. Site-specific glycan engineering has also been employed in therapeutic antibodies to install single glycans at a specific sequon to uncouple effector functions from antigen binding to create bifunctional antibodies in a single construct. In the vaccine field, site-specific glycan engineering can be used to create glycoconjugate vaccines with homogeneous antigen density on the carrier protein, with the potential to reduce the batch-to-batch variability that can make these vaccines challenging to manufacture and bring to market. Site-specific glycan engineering can also be used to create multivalent displays with multiple distinct glycan antigens at specific locations on a single carrier particle to increase breadth of protection with a single therapeutic platform. In addition, site-specific glycan engineering can be used to produce glycan-functionalized nanomaterials for diagnostics with defined multivalency for improved recognition of disease-associated carbohydrate biomarkers. The complexity of therapeutic proteins is also rapidly increasing, as they become optimized for complex biophysical and pharmacokinetic properties including half-life, tissue penetration, and immune activation. Site-specific glycan engineering will play an increasingly important role in being able to fine-tune multiple parameters of therapeutic proteins at once, since it allows for the design of glycans at specific sites on the protein.
Site-specific glycosylation is particularly useful for the development of therapeutic proteins and can be seen as a new approach for using glycans as a new design rule rather than as an unpredictable, often undesired, and uncontrolled post-translational modification (PTM). Antibodies are the first class of therapeutics that have adopted this new design rule as a major control parameter. By site-specifically adding an afucosylated glycan to the well-conserved Fc sequon of therapeutic antibodies, the affinity to Fc-γRIIIa is enhanced and ADCC is improved while Fab-induced antigen binding is unaffected, which would be much more challenging using changes to overall glycoforms since Fab and Fc are in close proximity. A further example of site-specific glycosylation use is the site-specific introduction of additional N-linked glycosylation sites in the Fab region to enhance the serum half-life of antibodies. The increase in the molecular weight and size of the antibodies leads to a slower rate of renal filtration and clearance, while the kinetics of target binding is unaffected. Site-specific glycan attachment can also be used to avoid immunogenicity by putting bulky, sialylated glycans at the site of a solvent-exposed non-human sequence. In this way, the offending sequence is sterically blocked from anti-drug antibody (ADA) recognition, while bioactivity is unaffected. Site-specific glycosylation also becomes important for biosimilar development, since the glycan occupancy at each sequon should be matched to the originator to ensure functional consistency and regulatory approval. Site-specific glycosylation has also been employed in enzyme replacement therapies (ERT) to specifically add mannose-6-phosphate groups that direct the ERT to the lysosome and in fusion proteins to help control their tissue penetration. Site-resolved addition of glycans can also be used to decouple the effect of sugars at different locations on specific functional parameters.
Site-specific glycan installation also allows for precise control over antigen display architecture, which is essential for the development of glycoconjugate vaccines. Conjugating carbohydrate antigens to protein carriers can generate immune memory, but chemically conjugated products are heterogeneous and often suffer from inconsistent antigen loading. Site-specific conjugation approaches, by contrast, use genetic fusions that place an exact number of polysaccharide antigens on each carrier protein. This yields well-defined vaccine products that ensure consistent antigen display from one manufacturing lot to the next. Engineered bacterial outer-membrane vesicle (OMV) vaccine platforms can be used to display site-specifically conjugated antigens. For instance, a SpyTag fusion to surface-exposed outer membrane proteins enables purified antigens to be site-specifically conjugated to SpyCatcher-decorated vesicles in vitro. Display of multiple epitopes on a single, multivalent vaccine carrier can be achieved without the need for a multi-expression system that could suffer from genetic instability over time. The result is a modular "click chemistry" platform, in which antigens can be expressed in the most suitable systems, folded properly, and then conjugated to the vaccine carrier. Site-specific glycoengineering can also be used in cancer immunotherapy to precisely control the display of tumor-associated carbohydrate antigens on protein scaffolds. By tethering antigens such as Lewis-X or Globo-H structures on proteins at defined densities, different spacing can be tested to determine what density and array of epitopes best activate T-cells. Site-specific glycan installation can also be used to target specific pattern recognition receptors. For example, polysaccharides from medicinal plants can be placed on vaccine carriers to specifically target TLR2-TLR6 heterodimers to direct a specific immune response. Site-specific modification can also be used to homogenize an antibody drug conjugate (ADC) payload site. Traditional ADCs can have a wide distribution of drug load, leading to subpotent or overdriven species. ADCs can be created by site-specific modification of a glycan in the Fc domain of an antibody to provide a single site for drug conjugation and defined drug-antibody ratios. In addition, the ease of site-specific re-engineering provides the flexibility needed for prototyping new antigens for pandemic preparedness.
Fig. 2 New therapeutic applications of N-linked and O-linked glycan modifications.2,5
Site-specific glycan engineering has transitioned to the clinic, moving beyond proof-of-principle, to become therapeutic platforms that offer solutions to key bottlenecks in current biopharmaceutical production approaches. Monoclonal antibodies have been some of the most immediate targets of this technology. For example, in oncology and metabolic disorders, the site-specific placement of a carbohydrate is directly correlated with therapeutic benefit. Antibody-drug conjugates (ADCs) have long suffered from chemically mediated heterogeneity and site-specific glycan engineering has been used to prove the concept of site-specific remodeling to homogeneity versus the inherently heterogeneous ADCs, which is accompanied by different pharmacokinetics (PK) and toxicity. Site-specific glycan engineering has been later applied to cytokines that have had a limited therapeutic index due to their short serum half-life. Site-specific installation of a complex glycan on defined positions have improved serum half-life without significantly impacting signal potency of the cytokines, thus widening the window of immunomodulatory therapeutics. Metabolic disorders have also benefitted from this technology, where site-specific engineering of insulin variants has resulted in fibrillization-resistant biologics without losing their activity in receptor-activation speed. These examples highlight the fact that site-specific glycan engineering is more than an orthogonal analytical tool, but a design feature that opens up avenues for rational optimization of a number of different performance attributes within a single protein scaffold. With increasing scrutiny on glycan heterogeneity from regulatory agencies, the ability to precisely define occupancy at a desired position is becoming a critical aspect of approval strategies, and site-specific glycan engineering is likely to become a commercial expectation rather than a novelty.
Site-specific glycan editing also allows for the separation of structure and function without redesigning the protein backbones. Site-specific modification of proteins for purposes such as antibody-drug conjugates (ADCs) is a highly desirable goal. Antibody-drug conjugates are expected to function stoichiometrically (i.e., each antibody has the same number of attached payload) to improve pharmacokinetics and facilitate modeling and dose optimization. Conjugation reactions that depend on random lysine acylation or cysteine alkylation produce heterogeneous mixtures that require extensive characterization. Site-specific modification of N-linked glycans in the Fc domain is an attractive solution as this region is separated from the antigen binding site, ensuring glycan editing does not impact antigen affinity, and as every antibody has an N-linked glycan at the Fc domain, site-specific derivatization of the Fc domain is straightforward. Glycan remodeling to chemoenzymatically trim each heterogeneous N-linked glycan down to a core GlcNAc, which is then modified by transglycosylation of an activated sugar oxazoline, can install a click-chemistry tag or payload with essentially 100% efficiency and thus generates a conjugate with defined stoichiometry (no subpotent or overmodified molecules). Site-specific glycan editing is also ideal for installing disease relevant mutations at the Fc domain or controlling glycan composition specifically at the Fc domain to address different mechanistic questions. For example, site-specific glycan editing can be used to silence Fc effector functions of immune checkpoint antibodies, such as anti-PD-L1 antibodies, to avoid depletion of activated T cells while maintaining antigen binding. Such antibodies are undergoing late-stage clinical trials and as site-specifically modified antibodies have been shown to be superior to stochastically conjugated antibodies, this is poised to become the standard for next generation biotherapeutics.
Site-specific modifications have been used to enhance the stability of therapeutic proteins, especially to prevent aggregation, fibrillization, or chemical degradation. For example, insulin in native form undergoes oligomerization and fibril formation during storage and injection. This can be avoided by the site-specific introduction of N-linked glycosylation sequons at conserved positions of either the A or B chain. The resulting glycosylated mutants self-associate much less and display no fibril formation, with no or minimal loss of signaling activity. Site-specific glycans prevent β-sheet fibril formation by steric and charge repulsion. The glycans are introduced at a specific site which is not in proximity to the receptor binding epitope, a condition which would not be met by random modification. The site-specific modifications are done in a chemoenzymatic manner: after expression of the aglycosylated form of the protein in bacteria, site-selective ligation is performed to add a GlcNAc-peptide tag to the N-terminus. Using engineered endoglycosidases, this tag can then be extended into complex, sialylated glycans. This generates homogeneous products, and differences in stability can be assigned to the defined glycan position, rather than the average effect of randomly distributed glycans. Site-specific glycosylation has also been applied to other cytokines such as interleukin-18 and interferon-α-2a to engineer more stable variants. In both cases, proteolytic degradation of the glycosylated proteins is prevented, which leads to longer serum half-lives due to higher molecular mass and sialic acid shielding. Importantly, by using a highly conserved glycosylation site, no new epitopes are formed by the glycan, since the peptide sequence of the glycosylation site recapitulates a naturally occurring glycoprotein.
Site-specific glycan engineering is challenging for several reasons. Glycosylation is a very elaborate process, and site-specific glycan engineering requires controlling glycosylation at multiple sites on a protein. This is difficult since engineered glycosylation sequons share a pool of nucleotide sugars and compete for the same processing enzymes in the Golgi apparatus, making the occupancy of each glycosylation site unpredictable. Furthermore, glycosyltransferases are very selective and have low tolerance to the peptide sequence flanking the glycosylation site, such that if a certain site is glycosylated, a different site on the same protein will not be. This is especially true for O-glycosylation as there is no specific sequon for this and the process is more dependent on polypeptide GalNAc-transferases and glycoprotein chaperones that are not well understood. Additionally, chemoenzymatic glycan remodeling is often not directly scalable from microgram scale to gram scale where low yield and side reactions can occur. Finally, as the glycosylation pattern of biologics is more and more regulated, new regulatory requirements are being put in place. Site resolved glycan mapping is now required for which the occupancy of each sequon is determined, calling for detection methods that are sensitive enough to detect trace under-glycosylated forms which may have altered PK/PD properties or increased immunogenicity. Site specific glycan engineering also currently remains relatively expensive as pure synthetic sugar donors, and engineered glycosyltransferases remain expensive at large scale. Resin immobilization is a possible solution but also requires validation of no enzyme leaching, and maintenance of activity through several cycles. Despite these challenges, new developments are making site-specific glycan engineering more widely accessible: AI-guided enzyme engineering is allowing for increased enzyme promiscuity, cell-free glycoprotein synthesis is being developed to avoid the toxicity issues of chemical glycan precursors in cells, and continuous manufacturing is allowing for in-line glycan analysis and process correction, all of which are putting site-specific glycan engineering on the path to become a more standardized biomanufacturing process.
This level of precision requires an integrated approach that takes into account the selectivity, efficiency, and analysis of the glycan addition at every step. The first precision barrier to overcome is the enzyme's selectivity, since the natural glycosyltransferases are only efficient and specific for certain peptide sequences and sugars. This is where protein engineering by directed evolution could be crucial, since random mutagenesis and selection of enzyme variants can broaden the specificities in a controllable manner for any bulky aglycones or new monosaccharides that the wild-type enzymes are not compatible with. The second precision barrier is the timing of the glycan addition, as adding a glycan too early in the folding process can result in misfolding of the protein. In this case, there is a need for light-activated or chemical-inducible glycosyltransferases that only add the glycan after the protein folds into its native conformation. In addition, spatial precision is required for selective glycosylation in the Golgi, since glycosyltransferases engineered to add specific glycans need to be targeted to the proper cisterna to find their substrates. For this, the glycosyltransferases can be fused to Golgi localization signals to direct them to the right subcompartments. Quality control also plays a critical role, as glycoproteomics by mass spectrometry (MS) needs to be sensitive enough to differentiate between fully occupied and partially modified sites, which may not be detected but can still impact the therapeutic performance. Recent advances in ion mobility-MS and electron-transfer dissociation can be utilized to resolve positional isomers and determine if a glycan is on the target sequon or another site with the same mass. The last precision metric is functional in that the added glycans need to match or improve the pharmacokinetics of the natural glycans without introducing new immunogenic epitopes, which will require a parallel assessment of both clearance mechanisms and immune recognition. Computational modeling to predict optimal glycosylation sites from structural data, and AI-based platforms to design enzyme-substrate pairs with minimal cross-reactivity, are being developed to get to the plug-and-play precision required for glycan installation on a range of therapeutic proteins.
High-precision glycan installation requires a holistic approach that tackles enzyme specificity, reaction kinetics, and detection at each glycosylation event. A major obstacle is the recognition of substrates by enzymes. Native glycosyltransferases (GTs) are highly specific for certain peptide sequences and sugar donors, which can be limiting when working with various protein backbones. Directed evolution of enzymes to relax but control their specificity could be a viable approach to accommodate large aglycones or rare sugars. Temporal specificity is another precision challenge; if glycosylation occurs too early, it may disrupt the protein folding process. Engineering light-activated or chemically inducible enzyme systems could allow for glycosylation to occur post-folding. Spatial precision is constrained by the localization within the Golgi; engineered GTs need to be retained in specific Golgi cisternae to reach their substrates. Fusion of GTs with Golgi localization signals can help target enzymes to the correct subcompartments. Quality control in terms of detection is crucial; the mass spectrometric glycoproteomics approaches need to be sensitive enough to differentiate between fully glycosylated and partially modified sites that may escape detection but can affect therapeutic efficacy. Ion mobility spectrometry and electron-transfer dissociation (ETD) allow for resolving positional isomers, providing insights on whether the glycan is attached at the desired sequon or an off-target site with the same mass. Functional precision, on the other hand, requires that the engineered glycans mimic or surpass the pharmacokinetic advantages of native glycans and do not introduce new immunogenic epitopes, which requires parallel assessment of clearance and immune recognition. As structure-based computational modeling starts to predict optimal glycosylation sites and AI-driven platforms start to design enzyme-substrate pairs with minimal cross-reactivity, we are getting closer to the precision required for plug-and-play glycan installation for all therapeutic proteins.
Achieve unmatched precision in protein design with our advanced site-specific glycan engineering solutions. Using cutting-edge enzymatic, chemoenzymatic, and genetic glycoengineering strategies, we precisely modify glycan structures at defined locations to enhance protein stability, activity, and therapeutic performance. Our workflows integrate high-resolution analytics—including LC-MS/MS, HILIC-HPLC, CE-MS, and site-specific glycoprofiling—to ensure every engineered glycan is accurately placed, fully characterized, and functionally validated. Our expertise enables you to:
Whether you are developing high-performance biologics or exploring novel protein engineering strategies, our site-specific glycan engineering solutions offer the precision, reproducibility, and scientific rigor needed to elevate your research and accelerate product success.
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