Solid-phase peptide synthesis (SPPS) involves the successive addition of protected amino acid derivatives to a growing peptide chain immobilized on a solid phase, including deprotection and washing steps to remove unreacted groups and also side products.

The ability to synthesize peptides to high purity in parallel and on a large scale has revolutionized research and also led to the development of a wide range of powerful therapeutic agents and vaccines. These applications require an almost limitless combination of sequences that often include modified amino acids, however, which makes considerable demands on synthesis optimization.

Let’s look at the advantages of using peptides as vaccines and therapeutics, how peptide modifications are helping to boost performance, and how this can be achieved with the right approach to SPPS, including chemistry and instrument design.

Tailored peptides facilitate rational vaccine design

Conventional vaccines have been powerful allies in fighting disease, but they have several drawbacks, including being limited to developing vaccines for organisms that can be grown in costly cell culture, low vaccine yields, additional unnecessary antigenic load that may induce unwanted responses, and the danger of non-virulent organisms converting to virulent forms.

These drawbacks have stimulated the development of peptide-based vaccines that offer the potential of providing, for example, fully-defined composition with no biological contamination, the possibility to customize, and cost-effective large-scale peptide vaccine production.

Peptide vaccines against infectious and chronic diseases

Peptide-based vaccines are under development against a number of pathogens, including the parasite causing malaria, hepatitis C virus, influenza virus, and HIV-1. Peptides are also featuring in the cutting-edge R&D efforts to fight the COVID-19 pandemic1. Another path involves a novel ’antidote-vaccine’ that could act as a prophylactic, immune-stimulant and therapeutic against the SARS-CoV-2 virus2.

Peptides are also being used in vaccines in cancer, in the form of neoantigens that are normally presented on the surface of tumor cells and can be targeted by T cells as foreign, leading to cancer cell death3. HER2-related peptides are being used to increase T-cell helper response in the fight against HER2-expressing tumor cells4. Alzheimer’s disease is also being addressed with peptide-based vaccines that target the aberrant aggregation of two proteins, Aβ and Tau4.

The art of mimicry – epitope choice and presentation

The immunogenicity of regions of antigens varies for both B- and T-cell epitopes. This immunodominance is particularly important for peptide vaccines that target only a single or a few critical epitopes. Choosing the most effective binding region or regions to focus on is therefore vital.

The peptide of a vaccine must also mimic the epitope presented by a structure that may be complex and conformation-dependent. Conformation-independent epitopes have low complexity and are formed by linear stretches of residues that may adopt local secondary structure once they are bound to the antibody. These linear epitopes are generally found in protein loops and are prime candidates for peptide vaccine design based on peptide synthesis.

Peptide customization to improve vaccine performance

While peptide-based vaccines can be effective and have a promising future, there are some disadvantages that must be addressed.

  • Poor immunogenicity – The simplicity of peptides becomes a disadvantage due to the limitations of the epitope that can be presented to the immune system. Short sequences can be added that are known to stimulate an immune response, or epitopes can be presented in multimeric formats based on virus-like particles (VLPs) or nanoparticles. Another common approach is the inclusion of immunostimulatory adjuvants.
  • Unstable in vivo – Peptide degradation can be reduced by binding to biopolymer conjugates or nanoparticles, which can also act as adjuvants.
  • Loss of native conformation – Peptide structure can be stabilized by adding flanking sequences, cyclization, stapling etc.
  • Effective for a limited population – The immunostimulatory power of peptide-based vaccines can be improved by multi-epitope constructs.

Peptides provide diversity in the search for new drugs

Therapeutic peptides have many advantages, including high activity, great chemical and biological diversity, and low toxicity. Added to that, advances in solid-phase peptide synthesis enable efficient synthesis of customized peptides with improved function and greater resistance to degradation, with high purity and at low cost compared to protein-based biologicals. These advantages are driving a dynamic peptide drug discovery process aimed at treating a range of conditions, including metabolic diseases, cancer, cardiovascular and infectious diseases, pain and hematological diseases.

Therapeutic peptides can function simply, in peptide replacement, such as insulin, or be used to expedite the delivery of cytotoxic substances or imaging agents into specific cells.

Examples of the diversity of research into therapeutic peptides include:

  • Spider venom peptides for non-opioid pain relief – the search for novel peptide analgesics that could specifically block ion channels involved in pain signaling5, 6.
  • Cyclotide scaffolds that stabilize epitopes – the design of cyclotides that help present epitopes in a constrained form that is also resistant to proteolysis7.
  • Epigenetic approaches to treating blood cancer –peptides designed to disrupt the binding of a chromatin modulator to its target8.

Therapeutic peptides can also function as antivirals, targeting HIV, influenza, or hepatitis, generally by inhibiting the replication cycle. This is one approach being pursued to tackle the ongoing COVID-19 pandemic, through the disruption of the interaction between the human angiotensin-converting enzyme 2 (ACE2) receptor and the viral spike protein of the SARS-CoV-2 virus responsible for this disease9.

Why are peptides used as vaccines and therapeutics?

Interest in peptide-based vaccines and therapeutics is increasing since peptides have a number of advantages that support a more rational design compared to more complex molecules and mixtures:

  • Fully-defined composition
  • Large-scale production is affordable
  • Water-soluble, stable in storage, can be freeze-dried
  • No biological contamination
  • Minimal allergic and autoimmune response
  • Can be customized
  • Different sequences can be quickly synthesized in parallel

The need to synthesize complex peptides

Success in the rational design, development and production of peptide vaccines and therapeutics depends on many factors, not least the ability to synthesize often complex peptides efficiently and quickly:

  • Longer peptides, >30 amino acids, are a challenge to synthesize, for example chemokines or histones.
  • Highly hydrophobic peptides such as beta-amyloids involved in Alzheimer’s disease and extensively used in research.
  • Cyclic peptides, in efforts to improve the rigidity, stability and resistance to degradation of therapeutic peptides. These are stapled, disulfide-bridged, bicyclic, or cyclized head-to-tail. Cyclotides, for example, are stable to proteolytic attack and have high thermal stability due to their highly constrained structure created by a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core.
  • Peptides can be branched, include side-chain modifications, phospho-peptides or be cysteine rich.
  • Post-translational modifications (PTMs) of native peptides may need to be mimicked to achieve maximum effect, for example in the case of neoantigens.
  • The need to maintain stereochemistry (risk of racemization during synthesis).

Purity is key

Progress in peptide-based academic research, and the development of therapeutics and vaccines relies on the optimization of SPPS. If every single step went to 100% completion, then only the full-length peptide would be produced. This is very rarely the case, however well a protocol is optimized. Side reactions and incomplete reactions even at a low level can soon result in low yield of the required product in a background of failed sequences and the resulting poor peptide purity that can affect the application.

For example, the theoretical yield of a 70-mer peptide can be calculated as follows:

  • 97% yield at every step (deprotection and coupling) gives 0.97140 = 1.4% overall yield

Increasing the efficiency towards 100% has dramatic effects:

  • 99% yield: 0.99140 = 24% overall yield
  • 99.5% yield: 0.995140 = 50% overall yield

Purity to match your application

The first thing to do is to decide what level of purity you require, or rather, what impurities may affect your application, e.g.:

Interference with target-binding

  • Deletion sequences
  • Truncation sequences
  • Incompletely deprotected sequences
  • Sequences modified during cleavage (reattachment of protecting groups at other locations on the peptide)
  • Other side-reaction products that are formed during synthesis (e.g. aspartimide formation, oxidation products)

Interference in cell-based assays

  • TFA (trifluoroacetic acid) – can kill cells used in a bioassay
  • Acetic acid

Avoiding these issues can require optimization or a look at downstream processing steps, such as including a salt exchange step. You may also need to examine the relative merits of freeze-dried vs. maintaining the peptide in solution, including stability testing.

A brief guide to purity goals

With this in mind, there are general guidelines to the purity you can aim at for a particular application:

  1. Crude (>50% or >70%). For high-throughput screening of lead compounds. >70% for an antigen when studying immune response, although specific contaminants may need to be removed.
  2. Medium purity (>70% or >80%). For the production, purification and testing of antibodies used in immunoassays, enzyme substrate studies, epitope mapping, affinity purification, bioassays or other immunological applications, and peptide screening.

    Medium purity >85%. For biochemistry and semi-quantitative applications, such as enzymology, epitope mapping, or studying biological activity, enzyme-substrate interactions, or phosphorylation, and also peptide blocking in Western blotting and cell attachment.
  3. High purity: >90% to >98% purity. For quantitative bioassays, quantitative in vitro receptor-ligand interaction studies, biological activity with ligand binding studies, quantitative blocking and competitive inhibition assays, quantitative phosphorylation and proteolysis studies, electrophoresis markers and chromatography standards.
  4. Extremely high purity: >98% purity. For in vivo studies, clinical trials, or drug studies that use peptides as pharmaceuticals, and structure-activity relationship studies. Protein structure analysis by nuclear magnetic resonance studies or crystallography.

How to optimize synthesis towards your required purity

Achieving the purity you need means optimizing the efficiency of correct coupling and reducing side reactions, deletions, racemization etc. to an acceptable level. This can involve addressing sequence-specific issues presented by, for example, long peptides (>30 amino acids), local sequences that cause aggregation, or complex peptides that include hydrophobic amino acids or modifications such as cyclic, or branched residues.

Optimization involves:

  • Sequence analysis, for example using software, to determine if there are regions that might present particular challenges in synthesis.
  • Assessing the risk for side reactions, such as racemization. Additional steps can reduce the risk of side reactions or help increase the efficiency of individual couplings, including pseudoprolines, dipeptide building blocks, additives, and alternative sidechain protection.
  • Method screening and optimization that includes key aspects of SPPS:
    • Resin selection – SPPS is normally conducted on polystyrene crosslinked with 1% divinylbenzene (DVB). Alternatives include polyethylene glycol (PEG) derivatives, and the choice of core can affect synthesis success and crude purity. Linkers attached to the bead provide a reversible linkage between the growing peptide and the solid phase support and determine the properties of the final product and the chemistry that can be used. The choice of linker determines the C-terminal functional group in the final product.
    • Coupling chemistry – Choice is generally dependent on the synthesis speed, with faster synthesis requiring more highly reactive (= unstable) coupling reagents (HCTU, HATU and COMU) than slower synthesis (DIC, HBTU). Reaction temperature can affect the choice of coupling chemistry.
    • Reaction temperature – Increasing the temperature may speed up synthesis and improve purity, but not always. Note that microwave-based heating is just heating and does not have a special effect on synthesis.
    • Monitoring deprotection by real-time ultraviolet monitoring – This can be important in optimizing protocols.
    • Double couplings – Difficult couplings can be handled by doubling coupling, extending the coupling time, or adding more equivalents.
    • Steric hindrance – Coupling reagents must be sufficiently rapid so that sterically hindered amino acids can be incorporated.
    • Capping – Permanently blocking any unreacted amino groups following a coupling reaction, or acetylating the N-terminus of a completed peptide can be useful for minimizing deletion products.
    • Fresh solvents – Re-using solvents reduces purity.

Peptide synthesizers for vaccines and therapeutic peptides

Optimizing the synthesis of complex peptides depends on handling complex chemistries while minimizing cross-contamination, dead volumes, and reagent carryover. This is especially important for the synthesis of long sequences, in which even small amounts of impurities, side products, and incomplete reactions over many cycles can drastically reduce the final purity and yield of desired peptides. Aggregation, secondary structure, steric hindrance, and conformational effects can still pose challenges in synthesis, and real-time deprotection monitoring optimizes reaction times to ensure complete deprotection.

In the case of neoantigen vaccines used in cancer therapy, parallel peptide synthesis is also important to quickly generate the pool of neoantigens needed. Speed is a critical factor since timing the delivery of a vaccine can make all the difference to the outcome. Neoantigens may be further altered through posttranslational modifications (PTMs) that occur in malignant but not healthy cells and are therefore an additional source of unique antigens that are specific to the individual patient.

Choosing a peptide synthesizer that will quickly deliver peptides with the right purity

A major factor that affects how you can optimize SPPS to achieve the peptide purity you need is the choice of synthesizer. Here are a few tips to help you in your choice:

  1. Parallel reaction vessels to screen methods and reagents — As we have seen, optimization can involve looking at a large number of variables. The ability to run different reagent combinations in parallel will greatly speed up the optimization process.
  2. Deprotection monitoring — Being able to monitor the efficiency of the deprotection step during the run avoids guesswork that can lead to incomplete deprotections, deletions, and side reactions.
  3. Temperature control — Increasing the temperature can improve purity for some sequences. Being able to test the effect of different temperatures in parallel can boost productivity.
  4. Minimal cross contamination — A peptide synthesizer that provides multi-channel synthesis with no reagent or resin cross-contamination will speed up optimization and minimize the risk of resynthesis
  5. No-prime single shots for rare monomers — Many of the monomers and reagents used in special chemistries can be expensive or precious, so some synthesizers offer prime-free ‘Single-Shot’ additions from any amino acid/monomer bottle position to ensure that nothing is wasted.
  6. Software designed for 21 CFR Part 11 compliance if peptides are being synthesized in a GxP facility.

You can find out more about designing a peptide synthesis and minimizing side reactions by downloading our recent summary of SPPS Tips for Success webinars:

  • Designing a Synthesis: How to maximize the likelihood of success when developing an automated synthesis protocol for a new peptide sequence.
  • Side Reactions: How to implement strategies to reduce the risk of unwanted side reactions that inevitably occur during the chemical synthesis process.
  • Peptide Manufacture: A real-world example of the synthesis of 24 SARS-CoV-2 peptides with therapeutic potential, manufactured with a focus on cGMP and regulatory compliance.


The ability to synthesize peptides to a high purity on a large scale, and also multiple sequences in parallel, has revolutionized many fields of basic research and also provided advanced weapons in the fight against disease. Gyros Protein Technologies provides you with the peptide synthesizers you need to match your needs in terms of yield, purity, scale, and synthesis in parallel.

You can find out more about how to choose a peptide synthesizer by reading the article ‘5 factors to consider when choosing a peptide synthesizer’


1 A candidate multi‑epitope vaccine against SARS‑CoV‑2. Kar, T et al. Scientific Reports. (2020) 10:10895.

2 Peptide antidotes to SARS-CoV-2 (COVID-19). Watson, A et al. bioRxiv, August 6, 2020. doi.

3 Personalized neoantigen vaccination with synthetic long peptides: recent advances and future perspectives. Chen, X et al. Theranostics 2020; 10(13): 6011-6023. doi: 10.7150/thno.38742

4 Peptide-based vaccines: Current progress and future challenges. Malonis, RJ et al. Chem. Rev. 2020, 120, 3210−3229.

5 Gating modifier toxins isolated from spider venom: Modulation of voltage-gated sodium channels and the role of lipid membranes. Agwa AJ et al, J Biol Chem. 2018 Jun 8;293(23):9041-9052. doi: 10.1074/jbc.RA118.002553. Epub 2018 Apr 27.

6 Webinar: Venom peptides: Rethinking voltage-gated sodium channel inhibition, Christina Schroeder

7 Targeted delivery of cyclotides via conjugation to a nanobody. Kwon S et al. ACS Chem. Biol., 2018, 13 (10), 2973–2980. DOI: 10.1021/acschembio.8b00653

8 Specific inhibition of DPY30 activity by ASH2L-derived peptides suppresses blood cancer cell growth. Shah, KK et al. Experimental Cell Research. Vol 382, Issue 2, 15 September 2019, 111485.

9 Evidence supporting the use of peptides and peptidomimetics as potential SARS-CoV-2 (COVID-19) therapeutics. VanPatten, S et al. Future medicinal chemistry. July 2020 DOI: 10.4155/fmc-2020-0180

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