Quality evaluation of synthetic quorum sensing peptides used in R&D

Abstract

Peptides are becoming an important class of molecules in the pharmaceutical field. Closely related peptide-impurities in peptides are inherent to the synthesis approach and have demonstrated to potentially mask biomedical experimental results. Quorum sensing peptides are attracting high interest in R&D and therefore a representative set of quorum sensing peptides, with a requested purity of at least 95.0%, was evaluated for their purity and nature of related impurities. In-house quality control (QC) revealed a large discrepancy between the purity levels as stated on the supplier׳s certificate of analysis and our QC results. By using our QC analysis flowchart, we demonstrated that only 44.0% of the peptides met the required purity. The main compound of one sample was even found to have a different structure compared to the desired peptide. We also found that the majority of the related impurities were lacking amino acid(s) in the desired peptide sequence. Relying on the certificates of analysis as provided by the supplier might have serious consequences for peptide research, and peptide-researchers should implement and maintain a thorough in-house QC.

Keywords: Impurity profiling; Quality; Quorum sensing peptides.

Fig. 1

(U)HPLC column flow chart for quorum sensing peptides analysis.

Fig. 2

Purity levels of quorum sensing peptides determined by in-house QC.

Fig. 3

The in-house QC control shows a distinct difference in peptide purity. (A) Chromatogram of supplier; Kromasil C₁₈-5 (250 mm×4.6 mm, 5 µm), quantification at 220 nm; (B) chromatogram of in-house QC; Vydac Everest C₁₈ column (250 mm×4.6 mm, 5 µm), quantification at 210 nm.

Fig. 4

TIC and UV chromatogram (A), MS¹ (B) and MS² (C) spectra of the related impurity C₁VFSLFKKC₉N (disulfide bond between C₁ and C₉) of peptide ID25 at a retention time of 23.47 min with appointed isotopic mass patterns and b- and y-fragmentation are allocated.

Fig. 5

TIC and UV chromatogram (A), MS¹ (B) and MS² (C) spectra of the related impurity LPFE(methyl)F of peptide ID134 at a retention time of 29.75 min with appointed isotopic mass patterns and b- and y-fragment allocation.

Fig. 6

TIC and UV chromatogram (A), MS¹ (B) and MS² (C) spectra of the related impurity TIPKY of peptide ID82 at a retention time of 9.59 min with appointed isotopic mass patterns and b- and y-fragment allocation.

Fig. 7

TIC and UV chromatogram (A), MS¹ (B) and MS² (C) spectra of the related impurity ILSGAPCIPWW of peptide ID121 at a retention time of 35.70 min with appointed isotopic mass patterns and b- and y-fragment allocation.

Fig. 8

TIC and UV chromatogram (A), MS¹ (B) and MS² (C) spectra of the related impurity ILSGAPCIPPPP of peptide ID121 at a retention time of 33.55 min with appointed isotopic mass patterns and b- and y-fragment allocation.

Fig. 9

Types of related peptide impurities found in the 61 experimentally determined sequences.