Custom Peptide Synthesis and their Purification Strategies

DATE: July 9, 2014

The synthesis of peptides to meet the modern clinical and therapeutic activities has been a challenge to organic chemists for almost a century. A wide range of advancements are continuously done to sustain new age requirements. Within these last three decades, a revolutionary transformation has been noticed in peptide chemistry world. New effective techniques in synthesis, purification, and analysis has made it a powerful tool in all fields of life sciences. Depending upon the structure, quantity or specific needs, the developers routinely perform difficult synthesis like synthesis of custom peptides, cyclic peptides, cosmetic peptides, etc.

All the latest technology and advanced solid phase techniques are utilized to make a standard peptide’s length range of 2 to 35mm, in quantities from 1mg to greater than 100mg. The peptides come in a standard range: unpurified, 70%, 80%, 90% and 95%. But it’s essential that users should know the quantity or purity level of the peptide before ordering it.

Purification Strategies

Purification is an essential step in Custom Peptide Synthesis, the crude sample only contains 40-50% purity level. The process used to produce peptides are optimized, but not 100% reliable. Reaction with free protecting groups or incomplete deprotection contains many by-products that are a result of deletion or truncated peptides, as well as the side products stemming from cleaved side chains or oxidation during the cleavage. The length of the peptide matters a lot in these events. The longer the peptide chain is, the more will be the probability of deletion or truncation.

The different strategies of peptide purification basically depend on the physiochemical features of peptides such as length, size, hydrophobicity and charge. The techniques are based on separation methods that include:

  • Partition chromatography
  • Size-exclusion chromatography
  • High-performance liquid chromatography (HPLC)
  • Ion exchange chromatography (IEC)
  • Reverse-phase chromatography (RPC)

HPLC is a traditional method of separation that captures the charged hydrophilic molecules in their stationary phase to give 100% purity. This is usually carried on with a reverse-phase or ion-exchange strategy to separate the peptides according to their hydrophobicity and charge interactions with the column materials. The amount of polar solvents are increased in their solvent phase, so that elution can be performed easily. In RPC, the C8, C4, C18 n-alkyl hydrocarbon ligands are captured by the stationary phase.

Purity of peptide is calculated by measuring the percentage of the target peptide with the impurities, obtained at (210-220nm) wavelength of peptide bonds. The purity levels vary as per the commercial requirement of the custom made peptides.

>70% – ELISA standards, ELISPOT assays and polyclonal antibody production
>80% – Maximum-throughput screening, Western blot analysing, antibody affinity purification, non-quantitative enzyme-substrate studies, non-quantitative blocking in immunohistochemical (IHC).
>95% – Quantitative studies such as NMR, receptor-ligand binding studies, ELISA and RIA, monoclonal antibody production, in vivo studies.

You only have to choose that company that satisfies the above mentioned purification strategies of peptide synthesis to yield a high end product. Order custom peptides from those companies only!

About The Author:

We synthesize virtually every antimicrobial peptide (AMP), such as LL-37, for Host Defense Peptide studies. Contact us today for pricing on your specific AMP of interest.