Critical parameters and checkpoints in the biochemical and biological QA/QC of synthetic peptides prior to use in assays of cell-mediated immunity

Stage of QC/QACommon problem(s)Proposed solution
Peptide set designDifficult to synthesize peptidesAvoid problematic C- and N-terminal residues where possible
Length and overlap of peptides15-mer-18-mer-length peptides with an overlap of 11-12 amino acids are optimal for CD4 and CD8 responses
Biochemical characterizationHPLC and MS reveal undesired impurities and side reaction productsResynthesize peptides using a realistic, cost-effective cutoff for desired purity; may need to redesign or even omit particular peptides
Dissolution and storagePeptide insolubility in aqueous buffers100% DMSO is the most universally applicable buffer for generic peptide solubility and is also compatible with downstream assay applications
StorageKeep all resolved peptides at −80°C and minimize freeze-thaw cycles
Biological characterizationPotential for inhibitory or stimulatory activity of individual or pooled peptides in downstream assay applications; this activity may not be predicted from the primary peptide sequence or from the biochemical analysis and requires empirical pretesting in the assay system of choiceScreen peptide pools in biological QA/QC assays using PBMC from as many as 50 subjects representing the HLA background in which the trial is to take place; screen for both inhibitory and stimulatory activity using the same assay to be implemented in the trial; deconvolute peptide pools to identify immunostimulatory or immunoinhibitory individual peptides; resynthesize and retest any problematic peptides