Peptide-based vaccines offer high specificity and an attractive safety profile but their overall clinical usefulness is primarily determined by the stability of the peptides. Manufactured or stored peptide antigens which break down before or after patient administration cannot properly expose their epitopes to APCs or B-cell receptors. The immune response may become ineffective when a vaccine loses structural fidelity because the protective neutralizing antibodies get replaced by non-neutralizing or tolerogenic antibodies. The breakdown of peptide structures can produce abnormal epitopes that activate inappropriate immune responses resulting in increased chances of adverse effects or autoimmune responses. Peptides with poor stability may necessitate cold-chain storage (2–8 °C or lower) and distribution conditions, inflating the manufacturing and logistical costs, limiting distribution in low-resource regions and leading to high vaccine wastage. In comparison, a peptide antigen that is chemically stable at room temperature following lyophilization can be ubiquitously accessible.
Peptide degradation is an unavoidable event that can occur via multiple, interconnected mechanisms, and these can be chemical (hydrolysis of backbone amide bonds, deamidation of Asn/Gln residues, side chain oxidation of Met, Cys or His residues), enzymatic or physical. Chemical processes are often accelerated by trace metal ions (Fe2+, Cu2+) or high pH. Elevated thermal stress also speeds up chemical reaction rates (e.g. aspartic acid–proline bonds are particularly sensitive to acid-catalysed cleavage at 37–45 °C). Disulfide bridges can be selectively reduced by β-elimination or scrambled during β-elimination/recombination at alkaline pH and elevated temperature. Photodegradation can occur on exposure to UV light, which can lead to the formation of reactive oxygen species that target Trp, Tyr, and Phe residues, causing local modification and changes in epitope recognition. Enzymatic degradation by serum or tissue proteases (aminopeptidases, chymotrypsin, elastase) can occur within minutes in aqueous solution or after injection, and is particularly rapid for flexible regions of peptides. Physical aggregation can occur during lyophilisation or reconstitution, mediated by either hydrophobic collapse or formation of β-sheet structures, leading to loss of native conformation and loss of immunogenicity. Inadequate formulation excipients (choice of sub-optimal buffer species such as phosphate vs. glutamate, insufficient quantity of anti-oxidants) can also influence degradation kinetics. To mitigate degradation, various strategies can be applied including cyclization, stapling, D-amino acid substitution, lyophilisation, and use of metal chelators or radical scavengers.
Degradation of peptide antigens can significantly alter their effect. Loss of conformational epitopes means that the peptides can no longer bind effectively to B-cell receptors, resulting in low-affinity antibodies that are likely not neutralizing. Fragments of the peptide may be rapidly cleared from the body by filtration in the kidneys, reducing the time antigens are present in the lymph nodes and resulting in a reduced ability to prime T-cells. Misfolded or aggregated peptides may result in processing into cryptic epitopes, which can result in a different immune response than expected and may provoke autoimmunity or an immunodominance hierarchy which suppresses the desired response. Binding to MHC class I and class II molecules may be reduced if the peptide has been degraded and no longer has a structure which allows for optimal binding to the groove. Activation of CD4+ T-cells and CD8+ T-cells would be reduced as a result. Additional dose would be needed to account for lost antigen during the course of administration, which could make it more costly and increase the potential for side effects. At 5–10 % degradation, the entire dose-response curve of a therapeutic can be shifted downward, resulting in potential failure to cross a protective threshold; early phase testing of a melanoma peptide vaccine showed this effect when stored at 4 °C. By contrast, cyclic or stapled analogues of peptides which do not degrade elicit high levels of neutralizing antibodies and polyfunctional T-cell responses after a single dose.
Peptides are widely used in various applications including immunology, medical diagnostics and drug discovery due to their high specificity and favorable safety profile. For their delivery as active pharmaceutical, delivery vectors or diagnostic imaging molecules, however, two main issues make their use challenging: their poor metabolic stability and their short half-life. Considerable efforts are currently being made to address those limitations, with structural modifications and novel delivery strategies being developed in order to enhance their capacity to reach their targets as active forms.
Fig.1 Strategies and tactics to improve peptide stability and delivery.1,2
Peptide destruction results from enzymatic cleavage, hydrolysis, oxidation, and conformational collapse. Chemical libraries encoding stability-enhancing modifications that do not interfere with peptide activity have been developed. Protease-sensitive sites can be substituted with un-natural amino acids (D-enantiomers, β-amino acids or α,α-dialkyl glycines) that prevent recognition by the enzyme, improving serum half-life 20-fold. Backbone amide N-methylation blocks endopeptidases and biases peptide conformations towards cis-amide structures that are more difficult for enzymes to cleave, and cyclosporine A, which contains seven N-methyl residues, demonstrates that such extensive modification can still result in oral bioavailability. Labile CO–NH peptide bonds can be replaced with peptide-bond isosteres (ψ[CH2NH] or retro-inverso bonds), and side-chain halogenation (fluoro-Phe, chloro-Tyr) can improve hydrophobic packing or oxidative resistance. Terminal capping groups (N-acetylation, C-amidation) can prevent diketopiperazine formation and exopeptidase attack. In vaccine applications, lipidation (C12–C18 fatty acid conjugation) or PEGylation can increase hydrodynamic radius, retard renal clearance, and sequester the peptide from proteolytic enzymes. Glycosylation or fluorescent tagging can be introduced using click chemistry to both stabilize the peptide and allow it to be tracked in vivo.
Cyclisation is a well-known, widely used and simple modification strategy for peptide drugs. It can confer several attractive properties to peptidomimetics and linear peptides, including improved target selectivity and specificity, increased cell permeability, and enhanced stability. Cyclic peptides exhibit a fixed geometric shape, lowering the entropy cost of binding and affording the ability to bind to their target with high efficiency and selectivity. In contrast, the high conformational flexibility of linear peptides can result in non-specific and off-target binding, increasing the probability of unwanted side effects. Cyclisation can take several forms, including backbone—backbone cyclisation, side chain—backbone cyclisation, and side chain—side chain cyclisation. Cyclisation is one of the most simple and universal single strategies to improve peptide stability. Cyclisation through covalent linkage of the N- and C-terminus or side chains (Lys–Asp lactam and Cys–Cys disulfide) eradicates free termini which serve as primary exoprotease attack sites and establishes a backbone low in entropy with resistance to proteases. Disulfide bridges act as a reversible conformational lock, which is ideal for extracellular targets, whereas thioether or hydrocarbon staples confer redox stability, desirable for intracellular delivery. RCM and Cu-free click cyclisation also enables the introduction of non-natural linkers, further rigidifying the scaffold, and can be used to increase membrane permeability. Quantitative stability assays show that cyclic analogues remain >90 % intact after 24 h in 90 % human serum, compared with<5 % for their linear counterparts. Critically, cyclisation can be tuned (via ring size and linker chemistry) to preserve, or even enhance, the native antigenic conformation required for strong immune recognition, and as such is a key strategy in modern peptide-vaccine design.
The peptide achieves stabilization from chemical and conformational modifications while protecting groups and formulation excipients safeguard it during synthesis storage and in vivo transit. Orthogonal protecting groups (Mtt or Dde for Lys, allyl for Asp/Glu, etc.) allow site-selective deprotection/cyclisation without having to subject the entire peptide to a more aggressive cleavage cocktail, thereby minimising side reactions. Freeze-drying peptides with trehalose or mannitol protects against aggregation. Citrate or histidine buffer (pH 5.5–6.5) suppresses deamidation, while metal chelators (EDTA or citrate) and anti-oxidants (ascorbate or methionine) are added to scavenge trace impurities that catalyze oxidation. Liposomal or polymeric nano-encapsulation is under development for injectable vaccines: The system acts as a protective micro-environment from proteases while providing slow-release capability which extends dendritic cells' antigen exposure time. Finally, single-use, nitrogen-flushed vials and desiccant-lined blister packs reduce moisture ingress and oxidative stress during global distribution to ensure the stabilised peptide retains its potency from factory to field. Hydrophobic ion-pairing (HIP) is a strategy that is currently being used to improve the stability of therapeutic peptides in aqueous solution. Hydrophobic ion pairing involves the use of ion pairs between a hydrophobic counterion and a positively charged amino acid residue in a peptide, typically His, Lys or Arg. The technique effectively masks the charged groups of the ion pair from the solvent, and reduces their exposure to water and potential hydrolysis. An advantage of the HIP approach is that it does not involve chemical modification of the peptide, which can alter the biological activity and potentially lead to undesirable side effects. Biodegradable arginine-based steroid-surfactants are used as cationic green agents for hydrophobic ion-pairing. The stabilizing effect of steroid-surfactant is shown by exposing model peptides to various stress conditions such as high temperature and low pH.
Linear peptide antigens are rapidly cleared by serum proteases, with many having half-lives of < 30 min, and this is often associated with poor antigen presentation. We overcome this weakness by pairing a modular cyclization toolkit with the size, hydrophobicity, and desired route of delivery of the epitope. Head-to-tail lactamization is the default cyclization strategy for 12–20-mer B-cell epitopes as it reproducibly gives 65–80 % isolated yield under microfluidic pseudo-dilution (1–2 mM) conditions to suppress intermolecular oligomerization. In epitopes with multiple cysteines, we resort to on-resin pairwise disulfide formation using orthogonal Acm/Trt protecting groups and controlled glutathione redox buffers to guarantee correct disulfide connectivity, which is key to maintaining native viral conformation. For larger constructs (>25 residues) or those with lipid tails, a CuAAC-mediated stapling installs a 1,2,3-triazole bridge that serves a dual purpose as a rigid turn inducer and a metabolically stable surrogate for an amide bond, increasing the serum half-life by more than 10-fold in mouse PK studies. Microwave-enhanced CuAAC (50 °C, 10 min) with tripodal amine chelators to suppress copper oxidation gives cyclization yields of >90 %, and click handles permit one-pot attachment of adjuvants (Pam3Cys, CpG) directly to the macrocycle. Bicyclic multi-epitope scaffolds - generated via sequential ring-closing metathesis and lactamization – display both conserved and variable viral epitopes without steric clash, and elicit neutralizing antibody titers that are 100-fold higher than that of linear peptide mixtures. All routes are locked by Design-of-Experiments (DoE) studies to ensure that the same cyclization parameters scale from milligram discovery lots to kilogram GMP campaigns.
A degraded vaccine candidate isn't just useless: it may shift the immune response to subdominant, non-protective epitopes. We mitigate potential failures through a three-step analytical process for our cyclic peptides which follows ICH Q7 standards and FDA vaccine regulations.
Peptide Synthesis Services at Creative Peptides
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