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Background and Significance <p>Racemization and deamidation impurities in therapeutic peptides represent critical quality attributes with profound implications for patient safety and therapeutic efficacy. These stereochemical degradations can alter peptide structure and clinical performance, necessitating comprehensive characterization and control strategies.</p> Objective <p>This investigation establishes a comprehensive analytical framework for systematic degradation assessment of cetrorelix acetate (CET), focusing on racemization and deamidation pathways.</p> Methods <p>A novel isocratic liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF) methodology was developed and validated according to ICH Q2(R2) guidelines. Systematic stress testing under hydrolytic, oxidative, thermal, and photolytic conditions was performed to generate degradation products, followed by advanced mass spectrometric characterization and computational toxicology assessment.</p> Results <p>The validated methodology demonstrated exceptional analytical performance with baseline resolution of CET and all degradation products (≥ 1.5), linearity (R<sup>2</sup> = 0.9999), and sensitivity (LOD: 0.18&#xa0;µg/mL, LOQ: 0.54&#xa0;µg/mL). CET exhibited selective hydrolytic instability, with alkaline conditions inducing 18.9% degradation compared to 5.8% under acidic stress, while thermal stress in solution state generated 1.4% degradation. Three major degradation pathways were identified: <span>l</span>-proline<sup>5</sup> racemization forming DP-I (RRT 0.89) predominantly under acidic conditions, <span>l</span>-serine<sup>4</sup> racemization generating DP-II (RRT 1.03) exclusively under alkaline conditions, and C-terminal deamidation producing DP-III (RRT 1.18) under both pH conditions.</p> Clinical Significance <p>Mechanistic elucidation revealed pH-dependent stereochemical transformations with potential implications for immunogenicity and therapeutic efficacy. Computational toxicology assessment indicated varying safety profiles among degradation products, with hepatotoxicity predictions ranging from 48.7 to 51.7% and consistently high respiratory toxicity concerns exceeding 97%.</p> Conclusions <p>This analytical methodology establishes an essential foundation for monitoring stereochemical integrity of CET throughout pharmaceutical development, manufacturing, and storage. The identified degradation pathways provide guidance for formulation optimization and potential risk assessment in therapeutic peptide development.</p>

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LC-QTOF Characterization of Racemization and Deamidation Impurities in Cetrorelix Acetate: A Comprehensive Forced Degradation Study with In Silico Toxicity Assessment

  • 期刊论文

  • 发表日期：

    2025年10月26日

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  • Shikha Patel,
  • Priti J Mehta,
  • Haidar Abbas Masi

摘要

Background and Significance

Racemization and deamidation impurities in therapeutic peptides represent critical quality attributes with profound implications for patient safety and therapeutic efficacy. These stereochemical degradations can alter peptide structure and clinical performance, necessitating comprehensive characterization and control strategies.

Objective

This investigation establishes a comprehensive analytical framework for systematic degradation assessment of cetrorelix acetate (CET), focusing on racemization and deamidation pathways.

Methods

A novel isocratic liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF) methodology was developed and validated according to ICH Q2(R2) guidelines. Systematic stress testing under hydrolytic, oxidative, thermal, and photolytic conditions was performed to generate degradation products, followed by advanced mass spectrometric characterization and computational toxicology assessment.

Results

The validated methodology demonstrated exceptional analytical performance with baseline resolution of CET and all degradation products (≥ 1.5), linearity (R2 = 0.9999), and sensitivity (LOD: 0.18 µg/mL, LOQ: 0.54 µg/mL). CET exhibited selective hydrolytic instability, with alkaline conditions inducing 18.9% degradation compared to 5.8% under acidic stress, while thermal stress in solution state generated 1.4% degradation. Three major degradation pathways were identified: l-proline5 racemization forming DP-I (RRT 0.89) predominantly under acidic conditions, l-serine4 racemization generating DP-II (RRT 1.03) exclusively under alkaline conditions, and C-terminal deamidation producing DP-III (RRT 1.18) under both pH conditions.

Clinical Significance

Mechanistic elucidation revealed pH-dependent stereochemical transformations with potential implications for immunogenicity and therapeutic efficacy. Computational toxicology assessment indicated varying safety profiles among degradation products, with hepatotoxicity predictions ranging from 48.7 to 51.7% and consistently high respiratory toxicity concerns exceeding 97%.

Conclusions

This analytical methodology establishes an essential foundation for monitoring stereochemical integrity of CET throughout pharmaceutical development, manufacturing, and storage. The identified degradation pathways provide guidance for formulation optimization and potential risk assessment in therapeutic peptide development.