Overcoming Key Challenges in Glycosylated Drug Development

The Role of Glycosylation in Drug Design and Function

(1) Why Glycosylation Matters in Biologics and Peptide Drugs

The glycosylation process stands as the most complex and common type of protein post-translational modification while creating significant protein diversity and fundamentally affecting protein functionality. Multiple glycan residues attached to proteins transform the proteome while various polysaccharides and oligosaccharides structures provide stereostructural protein variety. Glycosylation strongly influences protein solubility as well as stability and activity. The composition of glycans in calnexin and calreticulin chaperone proteins tracks the folding state of nascent polypeptides within the endoplasmic reticulum (ER). N-glycosylation was localized on the side of the conserved structural domains of hepcidin which could regulate calcineurin secretion and promote hepcidin maturation. While if N-glycosylation occurs in other parts of hepcidin, it misfolds and over time accumulates with calnexin. Glycans are expected to develop new non-invasive biomarkers and new methods for early diagnosis, risk prediction and treatment of cancer are urgently needed. Some of the most commonly used serum biomarkers in clinical practice for cancer diagnosis and monitoring prognosis are glycoproteins. Whether they are small molecule inhibitors of enzymes in the glycosylation process, or glycosylation-modified mAbs and new vaccines, these modalities have shown the great potential to target glycosylation for therapy development. In summary, glycosylation acts as molecular "switches" to provide more diverse functions to proteins or lipids, and play important roles in all aspects of life.

(2) Common Types of Glycans Used in Therapeutics

N-linked glycans: N-linked glycans are present in 15–25% of human IgG antibodies' variable domain (heavy chain variable domain (VH) or light chain variable domain (VL)) regions. These N-glycosylation sites encoded by the V-region genes (so-called Fab N-glycans) are a result of somatic hypermutation, because very few germline alleles carry N-glycosylation consensus sequences (NXS/T). In recent years, more and more evidences indicate that Fab N-glycans can influence antibody binding affinity. Several mechanisms on how N-glycan in antibody V-regions impacts epitope binding have been proposed, including the bulk size of N-glycan to fill out the space between the antigen epitope and the antibody paratope, charge–charge interaction between N-glycan sialic acids and the antigen, and through steric hinderance effects that affect the binding. The IgG4 subclass has the highest prevalence of V-region glycosylation (44% versus 11%–15% in other subclasses). IgE has a two-fold higher propensity for Fab glycans than IgA or IgG1, suggesting that elevated Fab glycosylation might be a hallmark of Th2-like responses. Massive amounts of autoantibodies isolated from rheumatoid arthritis and some B-cell lymphomas were found to be carrying Fab N-glycans. They are also present on human anti-idiotype autoantibodies to anti-TNF-α antibodies adalimumab and infliximab. Antibody binding affinity can decrease by up to three orders of magnitude when N-glycans are removed from the complementarity-determining regions (CDRs). The removal of N-glycan in the antigen-binding sites of a human IgG alloantibody, however, decreases its ability to neutralize factor VIII (FVIII) procoagulant activity without loss of binding affinity. It was proposed that its Fab glycan sterically hinders the interaction between FVIII and chaperone partner. Fab glycans in the framework or constant regions play additional roles in increasing antibody stability and in vivo half-life.

O-linked glycosylation: GalNAc-type-O-glycosylation of Ser/Thr is the most common type of O-linked glycosylation, which can be initiated by up to 20 different GALNTs, with a portion seemingly having protein-specific functions. For example, some of these enzymes are responsible for generating simple truncated O-linked glycans known as cancer-associated Tn antigens. GALNT3 uniquely modulates the processing site of FGF23 that regulates phosphate homeostasis. GALNT11 specifically modifies the low-density lipoprotein receptor-related receptor family and enhances ligand binding. GalNAc-type-O-glycosylation in recombinant TNFR:Fc fusion protein has a significant impact on its pharmacokinetics. O-glycosylation affects ADAM proteases, β1-adrenergic receptor activation, and atrial natriuretic peptide potency. O-glycans attached to neuropeptide Y and the glucagon family members modulate receptor activation properties and extend half-lives, demonstrating the importance of O-glycosylation in peptide hormones. O-Fucosylation and O-glucosylation stabilized the folding of EGF-like and thrombospondin type 1 repeat domains. Recently, a proteomic-based strategy uncovers that one-third of 279 classified peptide hormones carry O-glycans and that many of these identified O-glycosites are predicted to serve roles in proprotein processing, receptor interaction, biodistribution, and biostability. Since O-glycans can impact biotherapeutics in a number of ways, such as impacting pharmacokinetics, decreasing the binding affinity of peptide–antibody fusions, and unexpected O-glycosylation in antibody fusion linkers for manufacturing issues, understanding this old player for the biological systems could help to develop new tricks for biotherapeutics applications.

Fig. 1 Classification of major manifestations of glycosylation on proteins.^1,2

Top Challenges in Developing Glycosylated Drug Candidates

(1) Site-Specific Glycosylation of Peptides or Proteins

Site-specific glycosylation (SSG) is a major consideration in the design of glycosylated drug candidates. This modification involves attaching glycans to precise locations on a peptide or protein molecule. Glycans must attach to particular protein sites because these attachments optimize protein functionality or stability. SSG is hard to control for two reasons: the unavailability of direct glycosylation pathway control and the complexity of glycosylation. The exact placement of glycosylation sites on mAbs determines their functional efficacy and helps minimize potential immune system reactions. The manufacturing of site-specific glycosylated mAbs uses the production of engineered cell lines and enzymatic glycosylation. Nicotiana benthamiana plant leaf systems represent plant expression systems which incorporate glycan engineering functionality. Methods for site-specific enzymatic glycosylation can provide more fine-tuned control of both glycan type and site specificity. These methods may demand extra processing steps while needing substantial additional validation.

(2) Maintaining Glycan Stability During Formulation

Achieving glycan stability during formulation is a common challenge in the development of glycosylated drugs. Glycans can be sensitive to degradation or modification during the formulation process, which can impact the stability and efficacy of the drug product. To overcome this challenge, formulation conditions, such as pH, temperature, and storage conditions, need to be carefully optimized to ensure glycan stability. For instance, the use of stabilizers and protective agents can help maintain glycan integrity during formulation and storage. Developing robust analytical methods to monitor glycan stability is also critical for ensuring product consistency and quality.

Characterizing Glycoforms Across Batches

(1) Reproducibility in Chemical or Enzymatic Synthesis

Glycosylated drug candidate synthesis protocols require reproducibility to maintain product quality while meeting regulatory standards. Changes in synthesis conditions or enzyme function and substrate supply lead to varied glycosylation patterns and drug performance. Employing well-characterized enzymes together with optimized reaction conditions helps improve reproducibility. By utilizing glycosyltransferases with enhanced specificity and activity engineered through advanced methods researchers achieve uniform glycosylation results. The implementation of robust process controls alongside validation studies helps maintain consistent glycosylation patterns during the synthesis process.

(2) Regulatory Considerations Around Glycosylation Consistency

Regulatory authorities require glycosylated drug candidates to demonstrate batch-to-batch glycosylation consistency. This could necessitate the development of analytical methods and quality control approaches to ensure and demonstrate this glycosylation consistency. The degree of acceptable glycosylation variability and its measurement are described in regulatory guidances. Reference standards and validated analytical methods must be used to demonstrate glycosylation consistency as per the requirements of regulatory agencies. The regulatory guidance outlines the requirement to assess how glycosylation affects drug performance and immune responses. As a result, characterization studies and preclinical assessments must be conducted to demonstrate safety and consistency of glycosylated drug candidates.

How Our Custom Glycopeptide Services Help You Overcome These Barriers?

(1) Precise Glycan Attachment and Sequence Control

Our custom glycopeptide services can offer high site specificity for glycan attachment and sequence control to allow for the installation of glycans at desired positions on peptides or proteins. Correct glycan placement is important to optimize both function and stability of glycosylated drug candidates. Site-directed glycosylation enables us to add glycans to a target protein at the site of a specific amino acid. The technique delivers accurate glycan placement on proteins and achieves enhanced reproducibility by maintaining uniformity through multiple iterations that reduce variations in the process. Other methods of specific glycan attachment can also be used, such as engineered cell lines or enzymatic methods. Genetically engineered HEK293 and CHO cell lines can be used to make glycoproteins with more uniform glycosylation patterns. These engineered cell lines are chosen and optimized to produce glycoproteins with the desired glycan structures.

(2) Glycopeptide Libraries for SAR Studies

Our glycopeptide synthesis services also cover the production of glycopeptide libraries for SAR studies. Glycopeptide libraries are a collection of glycopeptides with different glycan structures and sequences. Screening a glycopeptide library will also identify which glycopeptide has the most favorable structure for a given application. Glycopeptide libraries can also be screened for the glycan structure that results in the highest binding affinity or desired biological activity. Our glycopeptide libraries are constructed to include a wide variety of glycan types, both N-linked and O-linked glycans. The glycan diversity in these libraries will provide the researcher the needed glycan structure diversity to perform extensive SAR studies. Custom library design services are also available.

(3) Analytical Support: LC-MS/MS, HILIC, NMR, HPAEC

We offer analytical support to help you guarantee quality and consistency of glycosylated drug candidates. Our experience with various analytical methods like LC-MS/MS, HILIC, NMR, and HPAEC allows us to comprehensively characterize glycopeptides for you. For that, LC-MS/MS in-gel digestion is a useful analytical approach to identify and quantify glycopeptides in order to obtain information about the glycan structure and site of attachment. HILIC separation can be performed prior to mass spectrometry in order to enrich glycopeptides and thereby increase sensitivity. The NMR spectra of the glycopeptide can be used to obtain information about the structural components of the glycan as well as the peptide and the correct conformation of the peptide and glycan. HPAEC is also a valuable tool to determine the composition and structure of glycans.

Peptide Synthesis Services at Creative Peptides

References

1. Image retrieved from Figure 1 " Classification of major manifestations of glycosylation on proteins," He M.; et al., used under [CC BY 4.0](https://creativecommons.org/licenses/by/4.0/). The original image was not modified.
2. He M.; et al. " Glycosylation: mechanisms, biological functions and clinical implications." Signal Transduction and Targeted Therapy, 2024, 9(1): 194.
3. Zhong X, D"Antona A M, Scarcelli J J, et al. New opportunities in glycan engineering for therapeutic proteins[J]. Antibodies, 2022, 11(1): 5. https://doi.org/10.3390/antib11010005.
4. Noborn F, Nilsson J, Larson G. Site-specific glycosylation of proteoglycans: A revisited frontier in proteoglycan research[J]. Matrix Biology, 2022, 111: 289-306. https://doi.org/10.1016/j.matbio.2022.07.002.
5. Reusch D, Tejada M L. Fc glycans of therapeutic antibodies as critical quality attributes[J]. Glycobiology, 2015, 25(12): 1325-1334. https://doi.org/10.1093/glycob/cwv065.