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.
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-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 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.
While peptide-based vaccines can be effective and have a promising future, there are some disadvantages that must be addressed.
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:
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.
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:
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:
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:
Increasing the efficiency towards 100% has dramatic effects:
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
Interference in cell-based assays
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.
With this in mind, there are general guidelines to the purity you can aim at for a particular application:
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.
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.
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:
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:
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