Chemical synthesis may be not reproducible in many cases. The following 4 aspects may affect the final results.
Peptide purity – This refers to the amount of correct peptide product relative to all other impurities, EXCEPT moisture, and is determined by analytical HPLC. Peptide purity is the most common variability, depending on what is obtained from the synthesizer or the purification step. It is important to remember that in some cases, peptide impurities with one or few amino acid deletions can still be active and contribute to the overall activity of the peptide. Variability decreases as the purity of the peptide increases.
Peptide content – This is determined by amino acid analysis and should not be confused with peptide purity. Peptide content refers to the percentage of all the peptides present relative to the rest of the non-peptide impurities, including moisture. This is also a big source of variability. Peptides can have anywhere from 10% to 90% water content, depending on the amino acid composition. Hydrophilic peptides have a tendency to bind more water. In addition, lyophilization conditions (original sample volume, solvent composition, sample load in the lyophilizer, length of lyophilization, etc.) can also affect the moisture content of the final product. Our standard lyophilization of at least 48 hours removes most of the free moisture, but there can still be differences in the amount of bound water especially in very hydrophilic peptides. It is for this reason that it is important for the end user to measure the peptide concentration in the peptide solutions, if possible, to ensure that peptide activities are expressed based on the same amounts of peptide.
Chemical transformation – During transit or storage, the peptide can undergo a series of chemical transformations that may lead to inactivity. Common examples are peptides containing Asp-Gly in their sequences that lead to iso-Asp formation (usually inactive), cross-linking of Cysteine containing peptides through the formation of disulfide bonds, oxidation of methionine to sulfoxides and sulfones, oxidation of tryptophan, pyroglutamate formation, etc.
Non-peptide impurities – Most peptides are purified in the presence of trifluoroacetic acid (TFA) in the solvent and the amount of residual TFA and TFA salts in the peptide are difficult to quantify. Free TFA can generally be removed after lyophilization for at least 48 hours, but TFA salts with the peptide or with other buffer ions may be difficult to completely remove. If TFA or TFA salts are suspected to cause problems in the experiment, the customer should request specific buffers be used in the final purification and lyophilization steps.