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Characterization of structurally related peptide impurities using HPLC-QTOF-MS/MS: application to Cbf-14, a novel antimicrobial peptide

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Abstract

Cbf-14 (RLLRKFFRKLKKSV), a designed antimicrobial peptide derived from the cathelicidin family, is effective against drug-resistant bacteria. Structurally related peptide impurities in peptide medicines probably have side effects or even toxicity, thus impurity profiling research during the entire production process is indispensable. In this study, a simple liquid chromatography–high-resolution mass spectrometry (LC-HRMS) method using a quadrupole time-of-flight (Q-TOF) mass spectrometer was developed for separation, identification, and characterization of structurally related peptide impurities in Cbf-14. A total of one process-related impurity and thirty-two degradation products were identified, and seven of them have been synthesized and confirmed. These impurities have not been declared in custom synthetic peptides. The degradation products were divided into five categories: fifteen Cbf-14 hydrolysates, five Cbf-14 isomers, four acetyl-Cbf-14 isomers, two aldimine derivatives, and six oxidized impurities. Combined with the peptide synthesis and the stress-testing studies, the origins and the formation mechanisms of these impurities were elucidated, which provides a unique insight for the follow-up quality study of Cbf-14 and other peptide products.

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Abbreviations

AMP:

Antimicrobial peptide

SPPS:

Solid-phase peptide synthesis

DIC:

N,N'-Diisopropylcarbodiimide

AIMAOPA:

5-[(Aminoiminomethyl)amino]-2-oxopentanoic acid

AIMG:

N-(Aminoiminomethyl)glutamine

DACA:

2-Amino-4,5-dihydro-1H-1,3-diazepine-7-carboxylic acid

PCA:

4,5-Dihydro-1H-pyrrole-2-carboxylic acid

GSA:

Glutamic γ-semialdehyde

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Acknowledgements

This work was supported by the National Science and Technology Major Project of the Ministry of Science and Technology of China (Grant No. 2019ZX09201001-004-0052).

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Authors and Affiliations

  1. Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 211198, China

    Yitong Huo, Kehui Xu, Yuting Lu, Taijun Hang & Min Song

  2. China National Narcotics Control Commission-China Pharmaceutical University Joint Laboratory On Key Technologies of Narcotics Control, Nanjing, 210009, China

    Yitong Huo, Kehui Xu, Yuting Lu, Taijun Hang & Min Song

  3. State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing, 211198, China

    Lingman Ma & Changlin Zhou

Authors
  1. Yitong Huo
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  2. Kehui Xu
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  4. Lingman Ma
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  5. Changlin Zhou
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  7. Min Song
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Contributions

Yitong Huo: methodology, investigation, formal analysis, writing the original draft; Kehui Xu: data curation, formal analysis, investigation; Yuting Lu: conceptualization; Lingman Ma: resources; Changlin Zhou: resources; Taijun Hang: supervision, writing—review and editing, project administration; Min Song: conceptualization, writing—review and editing, project administration.

Corresponding author

Correspondence to Min Song.

Ethics declarations

The experiments were conducted with synthetic Cbf-14 materials provided by the Department of Pharmaceutical Analysis of China Pharmaceutical University commercially sourced from GenScript (China).

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The authors declare no competing interests.

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Huo, Y., Xu, K., Lu, Y. et al. Characterization of structurally related peptide impurities using HPLC-QTOF-MS/MS: application to Cbf-14, a novel antimicrobial peptide. Anal Bioanal Chem 414, 6485–6495 (2022). https://doi.org/10.1007/s00216-022-04205-1

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