Performance of an rFC-based assay for endotoxinquantification in hyperimmune sera

Vigil Sanit Debate, Rio de Janeiro, 2025, v.13: e02447 | Published on: 23/10/2025

Authors

  • Ellen Gameiro Hilinski Núcleo de Ensaios Biológicos e de Segurança, Centro de Medicamentos, Cosméticos e Saneantes, Instituto Adolfo Lutz, São Paulo, SP, Brasil | Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil Author https://orcid.org/0000-0003-0045-2739
  • Daniela Dal Molim Ghisleni Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil Author https://orcid.org/0000-0002-4942-8513
  • Carla Lilian de Agostini Utescher Laboratório de Virologia, Instituto Butantan, São Paulo, SP, Brasil Author https://orcid.org/0000-0003-2382-2878
  • Wagner Quintilio Laboratório de Biotecnologia Viral, Instituto Butantan, São Paulo, SP, Brasil Author https://orcid.org/0000-0002-3382-8184
  • Terezinha de Jesus Andreoli Pinto1 Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil Author https://orcid.org/0000-0002-0238-8227

DOI:

https://doi.org/10.22239/2317-269X.02447

Keywords:

Recombinant Factor C, Endotoxins, Hyperimmune Serum, In Vitro Test

Abstract

Introduction: Endotoxin contamination poses a significant risk to the safety of pharmaceutical products, particularly those administered parenterally. Ensuring patient safety requires strict adherence to the regulatory standards for sterile and pyrogen-free pharmaceutical formulations. Objective: This study aimed to evaluate the feasibility of using the Recombinant Factor C (rFC) method, specifically Endolisa®, for detecting bacterial endotoxins in hyperimmune sera (pentavalent antibothropic, anti-rabies, and tetanus antitoxin). Method: Samples were spiked with endotoxin solutions at concentrations ranging from 0.05 to 10 EU/mL and analyzed using the rFC kit. The key performance parameters were thoroughly evaluated, such as specificity, detection and quantification limits, precision, accuracy, and linearity. Each concentration was tested a minimum of five times across six points. Results: The method demonstrated precision with relative standard deviations ranging from 3.5% to 19.0%, accuracy for endotoxin recovery between 94.0% and 134.0%, and linearity across the concentration range of 0.05 to 5 EU/mL for the three sera. The detection and quantification limits were established at 0.05 EU/mL. Conclusions: The results confirm that the rFC method provides accurate, precise, specific, and linear quantification of endotoxins in hyperimmune sera within the range of 0.05 to 5 EU/mL at 1:100 dilution. However, for samples spiked with 10 EU/mL (1:100 dilution), the method did not meet the compendial criteria.

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Author Biographies

  • Ellen Gameiro Hilinski, Núcleo de Ensaios Biológicos e de Segurança, Centro de Medicamentos, Cosméticos e Saneantes, Instituto Adolfo Lutz, São Paulo, SP, Brasil | Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil

    icone_lattes.png http://lattes.cnpq.br/0221469216077762

  • Daniela Dal Molim Ghisleni, Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil

    icone_lattes.png http://lattes.cnpq.br/0097556129317759

  • Carla Lilian de Agostini Utescher, Laboratório de Virologia, Instituto Butantan, São Paulo, SP, Brasil

    icone_lattes.png http://lattes.cnpq.br/0890539496821097

  • Wagner Quintilio, Laboratório de Biotecnologia Viral, Instituto Butantan, São Paulo, SP, Brasil
    icone_lattes.png http://lattes.cnpq.br/6919274764756010
  • Terezinha de Jesus Andreoli Pinto1, Departamento de Farmácia, Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP, Brasil

    icone_lattes.png http://lattes.cnpq.br/8115987312003585

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Published

2025-10-23

Data Availability Statement

The contents underlying the research text are contained in the submitted manuscript and in the doctoral thesis available at: https://repositorio.usp.br/item/003218090

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v. 13 (2025) | Continuous publication

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How to Cite

Performance of an rFC-based assay for endotoxinquantification in hyperimmune sera: Vigil Sanit Debate, Rio de Janeiro, 2025, v.13: e02447 | Published on: 23/10/2025. (2025). Health Surveillance under Debate: Society, Science & Technology , 13, 1-10. https://doi.org/10.22239/2317-269X.02447