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Analytical method development for synthetic peptide purity and impurities content by UHPLC – illustrated case study

Peptide-related impurities found in synthetic peptides may be process-related (deletion sequences, insertion impurities, isomers, truncated sequences, incomplete deprotection, by-products generated during synthesis or the final cleavage) or peptide-degradation products. The overall purity of a synthetic peptide (and hence the total impurity level) is related to the size of the peptide. A standard amino acid coupling cycle should be >99% efficient, meaning for a peptide containing 20 amino acids, an overall crude purity of >82% would be expected, or >74% for a peptide with 30 amino acids. The potential number of individual impurities observed in a crude peptide could theoretically be in the millions.

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