Glycoengineering, the rational design of protein glycosylation, is transforming the development, production, and control of biopharmaceuticals. It has moved glycans from being considered a mere "tag" to a fundamental therapeutic design feature that can influence the safety, efficacy, and quality of protein therapeutics, including monoclonal antibodies, replacement enzymes, and designer vaccines. Carbohydrate structures are now being harnessed as an orthogonal degree of design freedom, such that glycan identity, branching architecture, and terminal groups are no longer random decoration, but are instead engineered to specifically and predictably impact pharmacokinetic properties, immunogenicity, and function of the drug. Protein glycosylation was once seen as an unmodifiable, heterogenous aspect of protein therapeutics; however, through modern glycoengineering efforts based on combinatorial genetic engineering, targeted metabolic pathway engineering, and post-production glycan remodeling, glycosylation can now be tamed and made consistent. Glycan profile has also been more widely recognized as an important quality attribute (EQA) in the regulatory space, pushing further towards the development of strategies to fully control and connect glycan structure with function. This has led to increased implementation of glycoengineering strategies both upstream in the drug discovery process to screen/select for desired glyco-codes in lead candidates and throughout manufacturing to track glycan consistency for batch-to-batch manufacturing quality.
Glycoproteins are a majority class of biopharmaceuticals: Of the 164 approved protein therapeutics, about half are glycoproteins whose activity critically depends on their carbohydrates. Thus glycosylation control has become a requirement for development, as mixtures cannot ensure safety or efficacy. The first tenet of glycoengineering is that natural glycosylation is an emergent property of the kinetic availability of glycosyltransferases and protein transit times through the Golgi, and results in mixtures that are not conducive to reproducible and tightly controlled manufacture. Glycoengineering is the confluence of metabolic engineering, enzymology and process development that brings a rational approach to controlling glycosylation. Through elimination of competing pathways, provision of limiting precursors and trimming of glycans on the mature protein, the biosynthetic flow can be steered towards homogeneous and human-like glycoproteins. Glycoengineering is now a regulatory requirement, as it allows developers to meet agency guidelines for detailed glycan characterization and batch-to-batch consistency, and is thus involved from clone selection to commercial manufacturing. Glycoengineering has been most successfully applied to monoclonal antibodies, as their glycosylation can modulate immune effector functions and half-life with relatively small perturbations, and to enzyme replacement therapies, which require a particular glycan to direct the enzyme to the proper cell type. The field of complex therapeutics relies on glycoengineering as its primary technological foundation which translates glycobiology mechanism understanding into the production of consistently performing clinical medicines.
Biopharmaceuticals are a class of drugs that are produced using recombinant DNA technology, cell culture, or harvested from natural sources, such as proteins, antibodies, vaccines, and nucleic acid therapeutics. Glycoproteins make up nearly 50% of approved protein therapeutics. These macromolecules are characterized by the presence of one or more oligosaccharides covalently linked to asparagine or serine/threonine amino acid residues in the protein. This posttranslational modification has a critical impact on the behavior of the protein. As small molecule drugs are chemically synthesized and generally well-defined with established synthetic pathways, glycoproteins are biosynthesized in vivo inside a living cell, the glycosylation of which occurs by a template-free, enzyme-catalyzed process. This process is difficult to control and leads to a heterogeneous mixture of glycoforms. The nature of the heterogeneity is cell-line specific; for instance in mammalian cell lines, N-glycans are largely complex-type and generally non-immunogenic, while in yeast, high mannose type N-glycans are more common and will be cleared quickly, and in plant cell lines, xylose- and fucose-containing glycoproteins are known to be immunogenic. The clinical relevance of such differences is very important as the glycans affect the protein folding efficiency, the protein resistance to proteolytic degradation, receptor binding and serum half-life by interaction with clearance receptors. For this reason, biopharmaceutical companies need to consider glycosylation as an impurity that needs to be fully characterized and controlled rather than as an unwanted variation. Glycoengineering is the tool to achieve this, moving glycoprotein production from a "black box" biological process to a designable biomanufacturing process to provide well-defined therapeutics.
Glycans are key determinants for drug pharmacokinetics (PK), immunogenicity, and pharmacodynamics (PD), with impacts on a drug's success at all phases of development. Terminal sialic acids on N-glycans in the liver can prevent clearance through interaction with asialoglycoprotein receptors and improve PK and half-life, thereby lowering the dose requirements. This effect has been used in analogues of erythropoietin, as well as in the majority of marketed monoclonal antibodies (mAbs). High mannose glycans can promote clearance through the mannose receptor on macrophages. This can be useful for active targeting applications but is unfavorable for systemic mAbs. Immunogenicity is a key concern for therapeutic proteins and depends significantly on glycosylation. Presence of non-human glycans such as α-1,3-galactose or NGNA sialic acid can overcome B cell tolerance and lead to production of anti-drug antibodies (ADA). ADA not only neutralize the drug's activity but can also lead to adverse immune responses. Glycoengineering is used to humanize glycosylation of recombinant proteins through gene knockout and supplementation with metabolic intermediates, to reduce the immunogenic risk of biologics. Fc glycans also play an important role in determining antibody effector function, which is an important determinant of PD. In particular, core fucosylation can prevent interaction with FcγRIIIa. Removal of the core fucose leads to strongly enhanced ADCC, which has been used to generate antibodies with enhanced tumor cell killing activity. Bisecting GlcNAc on Fc glycans can also impact CDC and anti-inflammatory signaling, giving a range of effector functions that can be selected for a given indication. Glycans are also important in many other biologics; mannose-6-phosphate tags are essential for enzyme replacement therapies for lysosomal storage disorders, while glycan bulk can modulate tissue penetration in protein and peptide fusion constructs. Glycan characterization is now a requirement for regulatory approval and extensive characterization studies must be done to map different glycan structures to activity. The critical impact of glycans at all stages of drug development has made glycan control an essential aspect for the development of safe and effective biopharmaceuticals.
The toolbox of glycoengineering includes the genetic, metabolic, and enzymatic methods, which have been used to reprogram the stochastic nature of glycosylation during protein production into a well-defined process. The first genetic engineering of host cells involves CRISPR-mediated knock out of competing glycosyltransferases such as Fut8 for the removal of core fucose to increase antibody-dependent cellular cytotoxicity or overexpression of sialyltransferases for the addition of terminal sialic acids that could increase serum half-life by preventing recognition from liver clearance receptors. These cell lines are usually CHO or HEK293 cells that are modified to have stable integration and ensure that they can produce a consistent glycoform distribution over multiple manufacturing runs. The metabolic engineering approach involves the supplementation of the culture media with synthetic sugar precursors such as azidoacetylmannosamine or fluorinated fucose analogues, which could introduce bioorthogonal handles for post-production modification or competitively inhibit specific modification events, without any permanent genetic change, thereby providing a reversible method of process control. An additional level of precision is then added by the enzymatic remodeling performed after production, where endoglycosidases are used to trim the heterogeneous glycans to a uniform core that is then rebuilt by purified glycosyltransferases to add the desired structures from a supply of activated sugar nucleotides, which can include non-natural sugars or complex topologies that the cells themselves are not able to synthesize. The chemoenzymatic approach has been used extensively to convert commercially available proteins into improved versions without having to start from cell-line development. For quality control, analytical platforms such as mass spectrometry and high-performance liquid chromatography are usually run in parallel to ensure that the engineered glycoforms are within the specification ranges that were set during process validation. As regulatory expectations for biopharmaceutical production increase, these glycoengineering approaches are being embedded as part of a formal control strategy to ensure that the glycan modifications remain stable during scale-up and site transfers.
The role of N-linked glycosylation in mAb function and other aspects of immunity.1,5
Glycosylation is also used to improve the therapeutic performance of protein drugs. This can involve extensive remodeling of their glycan patterns to enhance the therapeutic profile, often by increasing certain desirable effects while reducing undesired liabilities. The most common application is antibody-dependent cell cytotoxicity, where removal of core fucose through knockout or inhibition increases the affinity to the activating Fc-γRIIIa receptor on NK cells, leading to improved lysis of tumor cells at the same dose. Improved sialylation can lead to an extended pharmacokinetic profile, by addition of terminal α-2,6-linked sialic acids that mask the underlying galactose residues and thus block recognition by asialoglycoprotein receptors in the liver, as well as reduce renal clearance, allowing for more stable plasma concentrations and the possibility of less frequent dosing. Reduction of immunogenicity can be targeted by humanizing glycan patterns, such as removal of antigens such as α-1,3-galactose and NGNA sialic acid that are often present in plant- and rodent-derived glycoproteins, and that can increase the risk of ADA responses, which is an important consideration for drugs that require repeated dosing over a long period of time. Increased consistency and control of glycosylation is important for biosimilars, which require the glycan fingerprint to be as close as possible to the reference product in order to achieve functional similarity. This requires orthogonal analytical testing across methods such as mass spectrometry, capillary electrophoresis, and lectin binding assays. Certain types of novel therapeutics depend on glycosylation, such as bispecific antibodies, which often require asymmetric Fc glycosylation to avoid mispairing between molecules, while maintaining Fc functions, as well as Fc-fusion proteins, which use glycan bulk to tune the rate of tissue penetration. Glycosylation is also important for therapeutic proteins outside of antibodies, such as the addition of mannose-6-phosphate tags to direct recombinant enzymes and clotting factors to lysosomes, or glycan shielding to improve the stability of cytokines to proteolysis.
The chemical biology study of homogeneous N-glycans.2,5
Site-specific glycan engineering is the ultimate expression of glycoengineering where instead of global engineering to the host-cell line, individual sites on the protein can be precisely engineered to control glycosylation occupancy and composition at each glycosylation site individually. Site-specific glycoengineering is important in precision medicine settings because some glycosylation sequons are more important than others and adding or removing N-linked glycosylation sites using site-directed mutagenesis can be used to selectively decorate or strip glycans from individual protein domains, leaving others unmodified. This strategy allows the creation of asymmetric glycoproteins and is commonly employed in monoclonal antibodies to add extra glycosylation sites to the Fab fragment to bulk up the molecule to have a longer half-life while not adding to the Fc effector function, or removal of the Fc glycan to completely abolish Fc-mediated effector functions for antibodies where an antagonist phenotype is required to block immune cell activation. Site-specific control of occupancy is also important to ensure the desired site is fully glycosylated as opposed to partially occupied. Site-specific chemoenzymatic remodeling strategies can also be used to install site-specific non-native glycans or modified glycans with "click" handles or diagnostic labels that can not be made by the cell itself. These site-specific engineering strategies are also important for precision medicine where a patient's individual glyco-profile may be used to determine what glycan structures would be optimal for a therapeutic. For example, patients with pre-existing anti-glycan antibodies can be given glycoengineered protein therapeutics lacking these glycan epitopes to avoid immune detection. Disease specific glycosylation motifs such as tumor-associated carbohydrate antigens can be targeted by using site-specific glycan engineering to make antibodies specific for the cancer glycans but not those found on normal tissue. The potential challenge of this strategy is that often mutations to add or remove glycosylation sites can create protein instability by introducing unpaired cysteines or exposing hydrophobic patches and can lead to aggregation. This has led to iterative structural modeling and screening approaches to engineer glycosylation sites without protein misfolding. However, as computational prediction of glycosylation efficiency from primary sequence improves and AI based platforms are developed to design optimal site patterns, site specific glycoengineering will become routine for patient-personalized protein biologics where the glycan pattern is matched to the individual's disease and immune profiles.
Glycoengineering is the scientific discipline of controlling and tailoring glycans on therapeutic proteins during cell culture manufacture. The glycoform of a protein is now known to play an integral role in dictating the safety and efficacy of biopharmaceuticals. Glycoengineering is based on the principle that glycans can be engineered to optimize specific properties of a drug product. In monoclonal antibodies (mAbs), glycoengineering can be used to potentiate the effect of a mAb without increasing the administered dose. In enzyme replacement therapies, the presence of particular glycans can be used to enhance the targeting of the enzyme to particular organs or tissues. Glycoengineering is also central to the development of biosimilars, as the glycan profile of a biosimilar is expected to match that of the reference product. Regulatory agencies also now require glycan profiles to be controlled, and certain glycoforms are considered "critical quality attributes" that must be demonstrated to be similar to the reference product. Glycoengineering can also be used to design novel drug products such as asymmetric bispecific antibodies or multi-valent glycoconjugates that are not possible using natural glycosylation pathways. It is now considered best practice to integrate glycan design and control into the earliest phases of drug development, selecting drug candidates and developing the cell culture process with the desired glycan in mind. Glycoengineering is thus a critical link between fundamental glycobiology and the production of biopharmaceuticals that have a consistent and predictable clinical response.
The ability to induce immunogenicity is one of the major disadvantages of biopharmaceuticals. Immunogenicity in this context refers to the ability of the protein to activate the human immune system through induction of anti-drug antibodies (ADA). This can lead to adverse side effects and/or the induction of an immune response that neutralizes the protein. Glycoengineering can help to avoid this problem through a variety of means that work to remove potential immunogenic epitopes and increase those which have a tolerogenic effect. The most common glycan-related immunogenicity is the inclusion of glycans with terminal sugars that are not found in humans, such as α-1,3-galactose or NGNA sialic acid residues that are commonly incorporated by plant and yeast systems, respectively. This non-self glycan is immunogenic and thus requires removal via humanization of the expression host or post-production enzymatic removal. Sialic acid supplementation results in terminal α-2,6-sialylation, which can inhibit activation of immune cells through binding to Siglec inhibitory receptors and thus reduce immunogenicity. This can be beneficial or detrimental depending on the protein; if effector functions are not needed or are problematic, protein variants that lack glycosylation at the N297 site can be introduced, eliminating the Fc glycan and its potential to interact with Fc receptors to activate immune cells. This does have downsides as the loss of the glycan can lead to increased protein aggregation as well as faster clearance from serum. Bisecting GlcNAc residues introduced by overexpression of GnT-III can also help to reduce activation through Fc-γ receptors and thus reduce immunogenicity. Removal of immunogenic peptide epitopes can be achieved through glycan placement that blocks access by antibodies over the relevant site. Each of these changes must be shown to be present at a level that would not be expected to be immunogenic in clinical batches as part of a regulatory submission.
Half-life extension and bioavailability are a major goal for glycoengineering. By this term we understand the extension of interval between two consecutive injections or infusion of the drug with the unchanged or better efficacy. Efficacy remains the same but the duration of its effect increases in this case. The first explanation for half-life extension is the increase in molecular mass with each added N-glycan; this has the effect of increasing the size of the protein above the renal clearance cutoff, therefore shifting elimination from the fast urinary excretion towards slower receptor-mediated clearance. The second point is the terminal sialylation. The presence of sialic acid can prevent recognition of galactose residues by the asialoglycoprotein receptor, and thus prevent rapid clearance from circulation. In the case of EPO and its analogues, additional glycosylation sites have been engineered in order to increase sialylation. This leads to proteins which remain in serum significantly longer than their aglycosylated parent forms. The downside is that affinity of the engineered protein to EPO receptor is decreased in many cases, but the improved pharmacokinetics of the protein often more than compensates for the decreased receptor affinity. The amount of fucosylation can also effect half-life; while core fucosylation has been shown to have a more pronounced effect on effector function, it can change the flexibility of the protein and therefore its interaction with the neonatal Fc receptor. Glycans attached to the O-linkage, such as on fusion proteins, can also have an effect on bioavailability, as they increase the hydrodynamic radius of the protein and shield it from renal clearance, but can also prevent rapid diffusion from injection sites into the blood stream and must be optimized with respect to the intended site of action. Glycan heterogeneity must also be taken into account, as under-sialylated glycoforms can be responsible for the majority of clearance, leading to bi-exponential or multi-exponential clearance. This can be addressed post-production with sialyltransferases under controlled conditions to provide a more uniform degree of terminal sialylation and ensure more consistent bioavailability. Glycans can also contribute to better bioavailability for proteins administered by subcutaneous injection. Electrostatic repulsion between the glycan chains and the components of the extracellular matrix can aid in movement from the injection site and into lymphatic capillaries.
Clinical application of glyco-engineered antibodies has advanced from proof-of-concept studies to early- and late-stage development. Glyco-engineering has matured to the point where it is recognized that carbohydrate remodeling of antibodies can positively or negatively impact the therapeutic index in both oncology and autoimmune diseases. The first proof-of-principle example used chemoenzymatic methods to remove heterogeneous Fc glycans and rebuild them with homogeneous G2 glycoforms. G2 glycoforms hexamerize on the cell surface to enhance complement-dependent cytotoxicity (CDC) but not affect antibody-dependent cellular cytotoxicity (ADCC), thereby demonstrating that a single linkage change can shift the primary mode of cell killing. This work was quickly followed up in the fucose context, where loss of core fucose by gene knockout in antibody-producing cell lines was found to consistently produce antibodies with improved NK cell activity and better tumor regression in patients who had failed existing therapies, showing that the mechanism of action is directly impacted by sugar changes rather than dose escalation. More recent examples have started to target both the Fab and Fc glycosylation sites. This is done by either removing potentially immunogenic variable-region glycans and replacing them with more tolerogenic glycans or by adding non-fucosylated Fc glycans, thereby creating molecules with reduced hypersensitivity and increased effector function. Overall, these advances have firmly established glyco-engineering as a required design layer rather than an add-on feature, and glycoform distribution is now considered an active substance specification and thus must be formally validated by regulatory authorities. The data so far have clearly shown that glycan changes can translate into statistically significant differences in progression-free survival and overall response rate, and it is becoming clear that glyco-engineering is a predictive design paradigm that can align a molecule's structure with its mechanism of action in the clinic.
Several glyco-engineered monoclonal antibodies (mAbs) have now entered the clinic and some are commercially approved. The distinction between such mAbs and their unedited progenitors, as well as the clinical benefit, is a direct result of glyco-engineering with respect to core fucosylation. The first strategy was to engineer cell lines in which the only fucosyltransferase gene was knocked out. This allowed the creation of a cell line that only produced afucosylated proteins, resulting in improved binding to the activating Fc receptors (FcR) and deeper clinical response in lymphoma compared to the parent fucosylated antibody. A second success story is the introduction of bisecting GlcNAc residues by overexpression of the responsible glycosyltransferase. This change not only altered effector functions but also increased clearance rates. This became a positive attribute for radio-immunoconjugates in which rapid serum clearance can reduce radiation dose to non-target organs. The full removal of Fc glycans has been successfully performed to produce antagonistic antibodies that can block signaling without the risk of engaging effector cells. This has the potential to avoid off-target cytotoxicity towards healthy tissue. There are also economic benefits for afucosylated glycoforms, which have a simpler analytical footprint and do not need as much characterization during quality control checks. Overall, these examples have demonstrated that modifications of glycans on therapeutic antibodies can be used to broaden therapeutic index, reduce potential immunogenicity and modulate pharmacokinetic characteristics without altering the protein backbone, allowing an accelerated regulatory pathway to approval for improved biologics.
A strategy known as combinatory glyco-engineering is also being used in the field of cancer immunotherapy to both improve anti-tumor activity and decrease toxicity. It is common for checkpoint inhibitors against inhibitory receptors to be manufactured with Fc mutations that abrogate effector functions, such as to avoid depletion of activated T-cells. However, it is also possible to precisely "dial in" the serum half-life with glyco-engineering without affecting the null phenotype, to allow for optimized timing with radiotherapy or other treatments. Bispecific T-cell engagers also use two glycan modifications on each of their Fc regions to both increase antibody-dependent cellular cytotoxicity (ADCC) towards tumor cells, while avoiding fratricide of T-cells. The latter is an important safety consideration. A number of these tumor-associated antigen (TAA) targeting candidates recently entered clinical trials and are showing objective responses in patients that have failed checkpoint blockade, highlighting the clinical impact of glycan engineering. It is also now becoming possible to combine glyco-engineering with site-specific conjugation of payloads. The engineered glycans can be modified with chemical handles that are stoichiometric attachment points for cytotoxic drugs, enabling homogeneous antibody-drug conjugates with fixed drug-to-antibody ratios (DAR) set by the occupancy of the glycan instead of random chemical conjugation. This strategy improves the therapeutic index of the resulting ADCs by allowing for consistent delivery of payloads without losing antigen-binding or introducing heterogeneity that is seen in conventional conjugation approaches. As the field now moves into late stage clinical development, glyco-engineering is well positioned to be a universal adaptor that can be used to link immune modulation, pharmacokinetics, and site-specific drug conjugation on a single therapeutic platform.
Manufacturing and regulatory considerations in glycoengineering merge in the glycan "footprint" control at the heart of product specifications. The manufacturing reproducibility issue is central since glycosylation is an emergent property of cell metabolism that is influenced by pH, pO2, nutrient limitations and trace element availability of the culture media, all of which may drift in a long-term bioreactor process. Such perturbations may "shift" the overall biosynthetic flux towards alternative glycan forms that are not within the specifications set at process development and validated at the process qualification stage. Regulatory expectations now place glycan distribution in the top rank of critical quality attributes, and require that process analytical technology (PAT) be in place to measure glycoform distribution on-line and in real time, with intervention capabilities to adjust the process if it tends to go outside specifications. For glycoprotein biosimilars, demonstration of glycan similarity with the reference product is only possible by direct comparison using the same analytical system, chromatographic gradients and conditions, including the same derivatization method, with little room for drift and adjustments over time. Therefore, analytical redundancy is required in the manufacturing process, for example simultaneous measurements using HILIC-FLD and mass spectrometry to eliminate any analytical bias when assessing differences in glycoform distributions between products. After approval, changes such as a change of manufacturing site, scale-up, or variation in raw materials also have to be shown to be comparable, with additional bridging studies to demonstrate stability of the engineered glycoform fingerprint, sometimes involving extensive statistical equivalence testing. This adds to the cost of control, requiring high resolution instrumentation, training of specialized personnel, and well characterized reference standards for calibration over the lifetime of the product. These investments are non-negotiable and sponsors have received complete response letters from regulatory agencies when their control strategy was judged to be lacking and additional glycan data were requested. Glycoengineering must be baked into quality systems very early in development and sustained during commercial supply.
The regulatory expectation for glycoengineering has been set by ICH guidelines Q5E and Q6B which require carbohydrate content, antennary profile, and glycosylation site(s) to be characterized "to the extent possible", leaving each sponsor with the task of clearly interpreting such a subjective statement or facing agency push back at study reviews and annual meetings. In the recent guidance document from FDA on the evaluation of immunogenicity for protein products, quantitative requirements for assuring non-human epitopes such as NGNA sialic acid and α-1,3-galactose at below the level of detection are clearly stated, given that these glyco-residues have the potential to elicit anti-drug antibodies through B-cell tolerance escape. The EMA guideline on monoclonal antibodies is more explicit in its monograph where the extent of mannosylation, galactosylation, fucosylation and sialylation are expected to be determined and the distribution of major glycan species (the G0, G1 and G2 forms for instance) to be reported with specification limits established on clinical lots. The stringency is further increased in biosimilar development where regulatory authorities are expected to see a stepwise demonstration of analytical similarity followed by clinical bridging studies that must be complemented with functional data in the presence of even minor glycan differences to support that safety and efficacy will not be affected. The emergence of the quality by design concept has had a major impact on the establishment of a design space for glycan attributes that needs to be demonstrated to assure the quality of the product. On the other hand, uncertainty in setting quantitative acceptance criteria remains for low abundance glycoforms that could be clinically relevant yet represent minor species. This has ultimately lead to the internal establishment of glycan reference standards that define the method and anchor validation which is required for comparison between sites as inter-laboratory studies have revealed high variability in the measurement of sialylation and antennary profiling using different methodologies. Sponsors are thus required to file complete analytical procedure sections that are part of the marketing authorization, and changes to the column, enzymes, or even gradient conditions post approval will require prior-approval supplements. This underscores the importance of platform robustness from the beginning.
The quality control (QC) of glycoengineered biologics involves a complex analytical approach. This strategy must account for both macro (site occupancy) and micro (glycoform) heterogeneity and have adequate precision to meet regulatory standards and ensure product safety. Identifying critical quality attributes (CQAs) for such biologics is vital, and this is often done through risk assessment early in development. This assessment considers the potential impact of glycan attributes like core fucosylation, sialic acid linkage, and high-mannose structures on functionality, including half-life, effector functions, and immunogenicity. Multiple orthogonal analytical techniques are required. High-Performance Liquid Chromatography (HPLC) methods like HILIC coupled with fluorescence detection (HILIC-FLD) are used for the quantification of fluorescently labeled glycans, offering insights based on polarity and branching. Mass spectrometry (MS) is utilized for precise mass confirmation and provides information on isomeric composition. Capillary electrophoresis is another orthogonal technique that separates molecules based on charge-to-size ratio, complementing chromatographic methods. Reference standards are critical for system suitability and establishing that resolution between crucial peaks is adequate. For instance, the resolution between afucosylated and fucosylated G0 forms may be a CQA. Sample preparation steps are a significant source of variation in glycan analysis. Techniques such as solvent precipitation, solid-phase extraction, and size-exclusion cleanup can lead to non-stoichiometric recovery of glycoforms, potentially distorting quantitative glycan profiles and obscuring the presence of low-abundance glycoforms. Automation of sample preparation is an emerging trend in glycoproteomic workflow. It significantly reduces sample preparation time while providing increased recovery and process robustness. Method validation is crucial to ensure the quantitative capture of both high- and low-abundance glycoforms. Quality by Design (QbD) principles are integral to the process. Acceptable ranges for each CQA must be established, typically informed by clinical experience. The inherent heterogeneity of glycoproteins presents challenges in glycan specification, as minor glycoforms can have significant functional implications, necessitating low limits of detection (LODs) rather than wide specifications. Inter-laboratory reproducibility can vary based on operator experience, instrumental platforms, and data interpretation algorithms. This can lead to discrepancies in results for the same sample across different labs, underscoring the need for standardized protocols and external validation, such as round-robin studies, to demonstrate analytical proficiency. Continuous manufacturing, in particular, raises the bar for glycan quality control. Continuous HILIC monitoring is required with in-line fraction diversion to discard out-of-spec material before final formulation.
Since the inception of glycoengineering, it has progressed from being a research niche to an emerging mainstream tool in biopharmaceutical research and development. The glycoengineering field has established glycans as an essential feature of protein therapeutics by shifting glycans from variables to parameters. The parameters are tunable, and the process can be controlled to be consistent so that glycans can be used to systematically and rationally optimize PK, immunogenicity, and potency of biopharmaceuticals. It has also been recognized in the field that glycans are a fundamental property of proteins by regulatory agencies deeming glycosylation to be a quality attribute (QA) of therapeutic proteins, including biologics and biosimilars, with glycosylation being designated as a key critical quality attribute (CQA) for biotherapeutics. The maturity of glycoengineering today can be ascribed to advancements in rational and precise glycans modifications, optimization of existing approaches, and the development of new approaches. Emerging tools including systems glycomics, in silico and synthetic biology-based glycan engineering, precision medicine approaches, next-generation cell lines, automation, continuous manufacturing, and real-time analytics, are anticipated to make glycoengineering more mainstream in the years to come. The continuing challenges, i.e. engineering complexity, lack of standardization in analytical techniques, and scalability of manufacturing processes, are all expected to be solved in an orderly fashion with the advancement of enabling technologies. The approval of glycoengineered mAbs and cancer immunotherapeutics has demonstrated glycoengineering proof-of-concept, which led to investments in glycoengineering platforms. Moving forward, we expect to see patient-stratified glycan optimization and continuous manufacturing that will change glycoengineering into a predictable, and consistent process.
Accelerate your biologics development pipeline with our specialized biopharmaceutical glycoengineering and glycan optimization services. We offer precision control over glycosylation to enhance therapeutic efficacy, stability, safety, and manufacturability. Using advanced enzymatic, chemoenzymatic, and cell-line engineering platforms—paired with high-resolution analytics such as LC-MS/MS, HILIC-HPLC, CE-MS, and site-specific glycan mapping—we deliver comprehensive solutions tailored to modern biopharmaceutical needs. Our expertise supports both early-stage discovery and late-stage product optimization, enabling you to:
Whether you're optimizing monoclonal antibodies, fusion proteins, enzymes, or novel biologic formats, our biopharmaceutical glycoengineering and glycan optimization services provide the scientific precision and reliability needed to enhance product quality and accelerate successful development.
References
