CUSTOM PEPTIDE SYNTHESIS

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Useful guidelines for working
with synthetic peptides

How to test the solubility of your custom synthetic peptide

The most commonly used method to test solubility is based on charge determination. For small peptides with up to 5 amino-acids, distilled water remains the first option. For other cases, you can refer to this guide:

1. Attribute -1 to each acidic residue (Asp / D, Glu / E) and to the terminal carboxylic acid. Then, assign +1 to each basic residues (Arg / R, Lys / K, His/h) and the terminal amine. Sum up both values to determine the overall charge of your peptide
2. If the overall charge value is positive, try to dissolve your peptide in water. In case the peptide does not dissolve, acidify your solution with an acetic acid solution (10 to 30%). Add TFA if acetic acid does not allow peptide dilution du sufficient concentration
3. In case the overall charge is negative and the peptide does not contain cysteine residues, try to dissolve your peptide in water. If the peptide does not dissolve, add ammonium hydroxide to obtain the desired concentration.
4. If the overall calculated charge is zero, the peptide can be diluted with organic solvents (methanol, ethanol, isopropanol or acetonitrile). A small amount of DMSO diluted with water can be used depending on final application. Specific care is requested for peptide containing cysteine, methionine or tryptophan residues as they are sensitive to oxidation. In these cases, replace DMSO by DMF.

How to design antigenic peptides for vaccines and antibody production

Synthetic antigenic peptides represent powerful tools for polyclonal or monoclonal antibody generation and as components of peptide vaccines. For these applications, peptides need to be designed with two properties in mind: antigenicity and immunogenicity.The first term is used to describe the ability of an antigen to interact with an antibody’s functional binding site, while the second term describes a peptide’s ability to elicit a humoral an/or cellular immune response. For effective vaccine and antibody production, peptides must have both properties.

Enhancing peptide antigenicity can be achieved by:

1. Choosing hydrophilic sequences: soluble regions have surface-exposed hydrophilic residues, more likely to elicit an immune response.
2. Ensuring high epitope accessibility: steric hindrance can hamper antibody-antigen interaction even in hydrophilic regions, it is essential to ensure the epitope can be easily accessed in the native protein.
3. Opting for an optimal peptide length: to maximize antigenicity, peptides should have between 10 and 20 AA residues. Short peptides (<10 AA) are unlikely to be bound by antibodies, while long peptides (>20 AA) are likely to adopt three-dimensional conformations that do not accurately mimic the structure of the native protein.

Peptide immunogenicity can be maximized by coupling synthetic peptides with carriers keeping the following recommendations in mind:

1. Peptide orientation: the peptide should always be presented in a similar manner than it would be presented in the native protein.
2. Nature of the carrier protein: the carrier protein often contains several epitopes able to elicit an immune response, thus, choosing the right carrier is of utmost importance to ensure the. KLH and BSA are the most used carrier proteins. KLH is the preferred molecule because of its large mass and complexity which elicit a much stronger immune response.

Solid-phase peptide synthesis has dominated the market for custom production in the last couple of decades. The method, initially developed in the 1950s, has matured into a technology that remains unparalleled in terms of automation, cost-effectiveness, scalability, lead times, and yields.

This method is traditionally carried out on a solid support in a stepwise manner from the C to the N terminus. Nα-protected amino acids are used to control the direction of the synthesis process and thus minimize side reactions. Today, solid-phase synthesis makes use of two major N-terminus protective groups: Boc (t-butyloxycarbonyl) and Fmoc (9-fluorenylmethoxycarbonyl).

In addition to these protective groups, permanent protection groups are often attached to side chains. These groups prevent unwanted branching and can withstand several cycles of chemical treatment during the synthesis process. They are only removed in the final stage of the process using strong acids. Benzyl (Bzl) and tert-butyl (tBu) are two of the most widely used side chain protection groups.

The stepwise synthesis of peptides is carried out as follows:

1. Deprotection: Nα-protective groups need to be removed to allow the addition of a new residue at the N-terminus. Deprotection agents used in this step depend on the nature of the protective groups. In this way, TFA (trifluoracetic acid) is used for Boc and piperidine for Fmoc-protected amino acids.
2. Coupling: the addition of a new amino acid residue at the N-terminus of a polypeptide chain requires the activation of the C-terminal carboxylic acid. Carbodiimides such as dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC) are widely used coupling reagents.
3. Cleavage: after several cycles of deprotection and coupling, all remaining protective groups need to be cleaved from the new polypeptide chain. Strong acids such as hydrogen fluoride (HF), hydrogen bromide (HBr) or trifluoromethane sulfonic acid (TFMSA) are used to cleave Boc and Bzl groups, while a relatively milder acid such as TFA is sufficient to cleave Fmoc and tBut groups. During this stage, the peptide chain is also separated from the solid support in order to be further purified.

The use of strong chemicals to produce synthetic peptides may offer a challenge when it comes to purification. To overcome this limitation, liquid-phase synthesis is often employed to achieve GMP-grade peptide production. Despite being significantly more time-consuming and leading to lower yields than solid-phase synthesis, liquid-phase methods are still sparingly used to produce highly pure short peptides (<10 AA) for some applications.

Both solid-phase and liquid-phase synthesis are chemical methods for linear peptide production. When large peptides with complex secondary or tertiary structures are required, recombinant expression is a much better alternative. However, despite being able a good method to produce long peptides, recombinant expression suffers from an important disadvantage – it is restricted to natural amino acids produced and processed by the host organism.

For this reason, when peptides with complex structures and unnatural amino acids need to be produced, a semisynthetic approach may be ideal.

Most efficient peptide purification methods

Despite the high efficiency of most peptide synthesis methods, many of these processes may still generate undesired impurities due to incomplete deprotection, unwanted reactions between free protecting groups, truncation and/or deletion of amino acids, isomers, and other side products.

Removal of these impurities is recurrently achieved by using one or several purification techniques:

• Size-exclusion chromatography
• Ion exchange chromatography (IEC)
• High-performance liquid chromatography (HPLC)
• Reverse-phase chromatography HPLC (RP-HPLC)
• Gel-filtration HPLC

Among these, RP-HPLC is the most widely used process of purification. Unlike conventional HPLC that separates products according to the concentration of polar solvents on the mobile phase, RP-HPLC captures hydrophobic molecules from aqueous solutions and releases them in function of their hydrophobicity. This makes it easier to separate correctly synthesized peptides from undesired impurities.