NC3Rs: National Centre for the Replacement Refinement & Reduction of Animals in Research

Bibliography to support the adoption of 3Rs approaches in biologics quality control testing

A collection of key peer-reviewed research papers providing the scientific evidence base and methodological details necessary to enable the biologicals community to apply 3Rs approaches for the quality control and batch release testing of biological products with confidence.

Providing evidence of the robustness and reliability of 3Rs approaches for quality control and batch release testing was highlighted during our stakeholder engagement activities as a key requirement for the biologicals community to apply these approaches confidently. This bibliography has been developed to help overcome the scientific and technical barriers that affect the uptake of 3Rs methods, enabling the more widespread adoption of novel testing strategies.

On this page

How to use the bibliography

The five sections below reflect the broad testing categories most commonly identified during the review of WHO testing guidelines. We have also included a ‘General’ section that provides the wider context for the 3Rs in quality control and batch release testing. 

  • Publications under each category can be accessed by clicking on the category name in the list of page contents above.
  • To return to the top of the page, use the up arrow in the grey circle in the bottom right corner of the page.
  • To search the bibliography more specifically, for example by product type (e.g. poliovirus) or specific test/assay (e.g. monocyte activation test), use the ‘Ctrl+F’ keyboard shortcut.

General support for taking a 3Rs approach

  1. Kleinschmidt-Doerr K et al. (2025). The Merck 3 Baskets approach for creating roadmaps to phase out animal testing. ALTEX Nov 28 10.14573/altex.2505201
  2. Feavers I et al. (2025). Conference report WHO informal consultation on the draft WHO Guideline on the phasing out of animal tests for the quality control of biological products. Biologicals 92: 101862 10.1016/j.biologicals.2025.101862
  3. Lilley E et al. (2025). Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals: Summary of NC3Rs final report to WHO Expert Committee for Biological Standardisation. Biologicals 89: 101778 10.1016/j.biologicals.2023.101778
  4. Lilley E et al. (2025). Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals: Reports from a series of NC3Rs stakeholder workshops. Biologicals 89: 101777 10.1016/j.biologicals.2023.101777
  5. Hoefnagel M et al, (2023). Rational arguments for regulatory acceptance of consistency testing: benefits of non-animal testing over in vivo release testing of vaccines. Expert Review of Vaccines 22(1): 369–377. doi: 10.1080/14760584.2023.2198601
  6. Lilley E et al. (2023). Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals: Responses from a survey of National Control Laboratories and National Regulatory Authorities. Biologicals 84: 101721 10.1016/j.biologicals.2023.101721
  7. Dierick J-F et al, (2022). The consistency approach for the substitution of in vivo testing for the quality control of established vaccines: practical considerations and progressive vision. Open Res Europe 2: 116. doi: 10.12688/openreseurope.15077.2
  8. Lilley E et al. (2022). Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals: Responses from a survey of vaccines and biological therapeutics manufacturers. Biologicals 81: 101660 10.1016/j.biologicals.2022.11.002
  9. Saint-Raymond A et al, (2022). Reliance is key to effective access and oversight of medical products in case of public health emergencies. Expert Rev Clin Pharmacol 15(7): 805-810. doi: 10.1080/17512433.2022.2088503
  10. Lilley E et al. (2021). Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals. Biologicals 74: 24-27 10.1016/j.biologicals.2021.10.002
  11. Akkermans A et al, (2020). Animal testing for vaccines. Implementing replacement, reduction and refinement: challenges and priorities. Biologicals 68: 92-107. doi: 10.1016/j.biologicals.2020.07.010
  12. Cuff P and Woods A (2019). Regulating Medicines in a Globalized World: The Need for Increased Reliance Among Regulators. National Academies Press (US). doi: 10.17226/25594.2020
  13. Uhlrich S et al, (2018). 3Rs in Quality Control of Human Vaccines: Opportunities and Barriers. In: Kojima, H., Seidle, T., Spielmann, H. (eds) Alternatives to Animal Testing. Springer, Singapore. doi: 10.1007/978-981-13-2447-5_10
  14. Bruysters M et al, (2017). Drivers and barriers in the consistency approach for vaccine batch release testing: Report of an international workshop. Biologicals 48: Pages 1-5. doi: 10.1016/j.biologicals.2017.06.006
  15. Schutte K et al, (2017). Modern science for better quality control of medicinal products "Towards global harmonization of 3Rs in biologicals": The report of an EPAA workshop. Biologicals  48: Pages 55-65. doi: 10.1016/j.biologicals.2017.05.006
  16. Hendriksen C et al, (2008). The consistency approach for the quality control of vaccines. Biologicals 36(1): 73-7. doi: 10.1016/j.biologicals.2007.05.002
  17. Halder M (2001). Three Rs potential in the development and quality control of immunobiologicals. Altex 18 Suppl 1: 13-47. PMID: 11854853

Pyrogenicity and endotoxin testing

  1. Burgmaiyier L et al (2025) Validation of the Monocyte Activation Test Demonstrating Equivalence to the Rabbit Pyrogen Test. Int. J. Mol. Sci. 26: 11136. doi: 10.3390/ijms262211136
  2. Yi S et al, (2025). Advancing Pyrogen Testing for Vaccines with Inherent Pyrogenicity: Development of a Novel Reporter Cell-Based Monocyte Activation Test (MAT). Vaccines 13(10):1009. doi: 10.3390/vaccines13101009
  3. Thurman T et al, (2023). Comparison of pyrogen assays by testing products exhibiting low endotoxin recovery. ALTEX 40(1):117-124. doi: 10.14573/altex.2202021
  4. Charton E (2022). European Pharmacopoeia Approach to Testing for Pyrogenicity. American Pharmaceutical Review July/August 2022. Link to article: https://www.americanpharmaceuticalreview.com
  5. Etna M et al, (2022). Optimization of the monocyte activation test for evaluating pyrogenicity of tick-borne encephalitis virus vaccine. ALTEX 37(4): 532-544. doi: 10.14573/altex.2002252
  6. Tindall B et al, (2021). Recombinant bacterial endotoxin testing: a proven solution. Bio Techniques 70(5): 290-300. doi: 10.2144/btn-2020-0165
  7. Hartung T (2021). Pyrogen testing revisited on occasion of the 25th anniversary of the whole blood monocyte activation test. ALTEX 38(1): 3-19. doi: 10.14573/altex.2101051
  8. Bolden et al, (2020). Currently Available Recombinant Alternatives to Horseshoe Crab Blood Lysates: Are They Comparable for the Detection of Environmental Bacterial Endotoxins? A Review. PDA J Pharm Sci Technol 274(5): 602-611. doi: 10.5731/pdajpst.2020.012187
  9. Gorman R (2020). Atlantic Horseshoe Crabs and Endotoxin Testing: Perspectives on Alternatives, sustainable Methods, and the 3Rs (Replacement, Reduction, and Refinement). Front Mar Sci 30(7): 1-11. doi: 10.3389%2Ffmars.2020.582132
  10. Piehler et al, (2020). Comparison of LAL and rFC Assays-Participation in a Proficiency Test Program between 2014 and 2019. Microorganisms 8(3):418 doi: 10.3390/microorganisms8030418
  11. Vipond C et al, (2019). Development and validation of a monocyte activation test for the control/safety testing of an OMV-based meningococcal B vaccine. Vaccine 37(29): 3747-3753. doi: 10.1016/j.vaccine.2018.06.038
  12. Kirfalusi-Gannon J et al, (2018). The Role of Horseshoe Crabs in the Biomedical Industry and Recent Trends Impacting Species Sstainability. Front Mar Sci 05: 1-13. doi: 10.3389/fmars.2018.00185
  13. Bolden J and Smith K (2017). Application of Recombinant Facor C Reagent for the Detection of Bacterial Endotoxins in Pharmaceutical Products. PDA J Pharm Sci Technol 71(5): 405-412 doi: 10.5731/pdajpst.2017.007849
  14. Fennrich S et al, (2016). More than 70 years of pyrogen detection: Current state and future perspectives. Altern Lab Anim 44(3): 239-53. doi: 10.1177/026119291604400305
  15. Schindler S et al, (2009). Development, validation and applications of the monocyte activation test for pyrogens based on human whole blood. ALTEX 26(4): 265-277. doi: 10.14573/altex.2009.4.265
  16. Ding J and Ho B (2001). A new era in pyrogen testing. Trends Biotechnol 19(8): 277-81. doi: 10.1016/s0167-7799(01)01694-8

Neurovirulence testing

  1. Konz J et al (2021). Evaluation and validation of next-generation sequencing to support lot release for a novel type 2 oral poliovirus vaccine. Vaccine X 8: 100102 doi: 10.1016/j.jvacx.2021.100102
  2. May Fulton C and Bailey W (2021). Live Viral Vaccine Neurovirulence Screening: Current and Future Models. Vaccines (Basel) 9(7):710. doi: 10.3390/vaccines9070710
  3. Charlton B et al, (2020). The Use of Next-Generation Sequencing for the Quality Control of Live-Attenuated Polio Vaccines. J Infect Dis 222(11):1920-1927. doi: 10.1093/infdis/jiaa299
  4. da Costa A et al, (2018). Innovative in cellulo method as an alternative to in vivo neurovirulence test for the characterization and quality control of human live Yellow Fever virus. Biologicals 53:19-29. doi: 10.1016/j.biologicals.2018.03.004
  5. Wood D (1999). Neurovirulence. Dev Biol Stand 101:127-9. PMID: 10566785
  6. Horie H et al, (1998). Estimation of the neurovirulence of poliovirus by non-radioisotope molecular analysis to quantify genomic changes. Biologicals 26(4): 289-97. doi: 10.1006/biol.1998.0159
  7. Chumakov K et al, (1993). Assessment of the viral RNA sequence heterogeneity for control of OPV neurovirulence. Dev Biol Stand 78:79-89. PMID: 8388834

Adventitious agents testing

  1. Chin P et al (2025). Evaluation of high-throughput sequencing for replacing the conventional adventitious virus detection assays used for biologics. npj Vaccines (2025). doi: 10.1038/s41541-025-01351-2
  2. Alston A et al (2025). Validation of a Next Generation Sequencing Method for adventitious agents detection in a live vaccine matrix. Biologicals
    Volume 90, 101828. doi: 10.1016/j.biologicals.2025.101828
  3. Khan A et al, (2023). Report of the third international conference on next generation sequencing for adventitious virus detection in biologics for humans and animals. Biologicals 83: 101696. doi: 10.1016/j.biologicals.2023.101696
  4. Barone P et al, (2023). Historical evaluation of the in vivo adventitious virus test and its potential for replacement with next generation sequencing (NGS). Biologicals 81:101661. doi: 10.1016/j.biologicals.2022.11.003
  5. Khan A et al, (2023). IABS/DCVMN webinar on next generation sequencing. Biologicals 81: 101662. doi: 10.1016/j.biologicals.2022.12.001
  6. Morris C et al, (2021). Adventitious agent detection methods in bio-pharmaceutical applications with a focus on viruses, bacteria, and mycoplasma. Curr Opin Biotechnol 71:105-114. doi: 10.1016/j.copbio.2021.06.027
  7. Charlebois R et al, (2020). Sensitivity and breadth of detection of high-throughput sequencing for adventitious virus detection. npj Vaccines 5: 61. doi: 10.1038/s41541-020-0207-4
  8. Khan A et al, (2020). Report of the second international conference on next generation sequencing for adventitious virus detection in biologics for humans and animals. Biologicals 67: 94-111. doi: 10.1016/j.biologicals.2020.06.002
  9. Pennings J et al, (2020). A next-generation sequencing based method for determining genetic stability in Clostridium tetani vaccine strains. Biologicals 64: 10-14. doi: 10.1016/j.biologicals.2020.02.001
  10. Khan A et al, (2018). Report of the second international conference on next generation sequencing for adventitious virus detection in biologics for humans and animals. Biologicals 55: 1-16. doi:10.1016/j.biologicals.2018.08.002
  11. Khan A et al, (2017). A Multicenter Study To Evaluate the Performance of High-Throughput Sequencing for Virus Detection. mSphere 2(5).e00307-17 doi: 10.1128/msphere.00307-17
  12. Gombold J et al, (2014). Systematic evaluation of in vitro and in vivo adventitious virus assays for the detection of viral contamination of cell banks and biological products. Vaccine 32(24): 2916-26. doi: 10.1016/j.vaccine.2014.02.021
  13. Arifa S. Khan et al (2025). Report of the fourth conference on next-generation sequencing (NGS) for adventitious virus detection in biologics for humans and animals: Validation and implementation of NGS. Biologicals (92) doi: 10.1016/j.biologicals.2025.101859

Potency testing

  1. Iwaki M et al, (2023). An ELISA system for tetanus toxoid potency tests: An alternative to lethal challenge. Biologicals 82:101681 doi: 10.1016/j.biologicals.2023.101681
  2. Tedcastle R et al, (2022). Report on the WHO collaborative study to establish Universal Reagents for the D-Antigen potency testing of Inactivated Polio Vaccines. Link to article: https://www.who.int/publications/m/item/who-bs-2022.2432
  3. Vandebriel R et al, (2022). Development of a cell line-based in vitro assay for assessment of Diphtheria, Tetanus and acellular Pertussis (DTaP)-induced inflammasome activation. Vaccine 40(38): 5601-5607. doi: 10.1016/j.vaccine.2022.08.022
  4. Riches-Duit R et al, (2021). Characterisation of diphtheria monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay. Biologicals. 69: 38-48. doi: 10.1016/j.biologicals.2020.12.002
  5. Riches-Duit R et al, (2021). Characterisation of tetanus monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay. Biologicals. 71: 31-41. doi: 10.1016/j.biologicals.2021.04.002
  6. Signorazzi A et al, (2021). In vitro assessment of tick-borne encephalitis vaccine: Suitable human cell platforms and potential biomarkers. ALTEX 38(3): 431-441. doi: 10.14573/altex.2010081
  7. Stalpers C et al, (2021). Variability of in vivo potency tests of Diphtheria, Tetanus and acellular Pertussis (DTaP) vaccines. Vaccine. 39(18): 2506-2516. doi: 10.1016/j.vaccine.2021.03.078
  8. Ticha O et al, (2021). A cell-based in vitro assay for testing of immunological integrity of Tetanus toxoid vaccine antigen. NPJ Vaccines 6: 88. 10.1038%2Fs41541-021-00344-1
  9. Imura A et al, (2020). Development of an Enterovirus 71 Vaccine Efficacy Test Using Human Scavenger Receptor B2 Transgenic Mice. J Virol 94(6):e01921-19. doi: 10.1128/jvi.01921-19
  10. Kouiavskaia D et al, (2020). Universal ELISA for quantification of D-antigen in inactivated poliovirus vaccines. Journal of Virological Methods 276: 113785. doi: 10.1016/j.jviromet.2019.113785
  11. Michiels T et al, (2020). Degradomics-Based Analysis of Tetanus Toxoids as a Quality Control Assay. Vaccines  8(4): 712. doi:10.3390/vaccines8040712
  12. Rajam G et al, (2019). Development and validation of a robust multiplex serological assay to quantify antibodies specific to pertussis antigens. Biologicals 57: 9-20. doi: 10.1016/j.biologicals.2018.11.001
  13. Morgeaux S et al, (2018). Replacement of in vivo human rabies vaccine potency testing by in vitro glycoprotein quantification using ELISA - Results of an international collaborative study. Vaccine 35(6): 966-971. doi: 10.1016/j.vaccine.2016.12.039
  14. Chabaud-Riou M et al, (2017). G-protein based ELISA as a potency test for rabies vaccines. Biologicals 46: 124-129. doi: 10.1016/j.biologicals.2017.02.002
  15. Byung-Chul K et al, (2016). A collaborative study of an alternative in vitro potency assay for the Japanese encephalitis vaccine. Virus Res 223: 190-196. doi: 10.1016/j.virusres.2016.07.012
  16. Chacón F et al, (2015). The lethality test used for estimating the potency of antivenoms against Bothrops asper snake venom: pathophysiological mechanisms, prophylactic analgesia, and a surrogate in vitro assay. Toxicon 93: 41-50. doi: 10.1016/j.toxicon.2014.11.223
  17. Ma S et al, (2014). Development of a sandwich ELISA for the quantification of enterovirus 71. Cytotechnology 66(3): 413–418. doi: 10.1007%2Fs10616-013-9588-9
  18. Morgeaux S et al, (2013). Validation of a new ELISA method for in vitro potency testing of hepatitis A vaccines. Pharmeur Bio Sci Notes 2013: 64-92. PMID: 24447723
  19. He F et al, (2013). Development of a sensitive and specific epitope-blocking ELISA for universal detection of antibodies to human enterovirus 71 strains. PLoS One  8(1):e55517. doi: 10.1371/journal.pone.0055517
  20. Descamps J et al, (2011). A case study of development, validation, and acceptance of a non-animal method for assessing human vaccine potency. Procedia in Vaccinology  5: 184-191. doi: 10.1016/j.provac.2011.10.018
  21. Poirier B et al, (2010). Would an in vitro ELISA test be a suitable alternative potency method to the in vivo immunogenicity assay commonly used in the context of international Hepatitis A vaccines batch release? Vaccine 28(7): 1796-1802. doi: 10.1016/j.vaccine.2009.12.006
  22. Kumar S et al, (2009). Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum. Biologicals 37(5): 297-305, doi: 10.1016/j.biologicals.2009.05.002
  23. von Hunolstein C et al, (2008). Evaluation of two serological methods for potency testing of whole cell pertussis vaccines. Pharmeuropa Bio. 2008(1): 7-18. PMID: 19220977
  24. Winsnes R et al, (2006). Collaborative Study for the Validation of Serological Methods for Potency Testing of Diphtheria Toxoid Vaccines - Part 2. Link to article: https://www.edqm.eu/documents/52006/123862/bsp034-p2-dserol.pdf
  25. Shank-Retzlaff M et al, (2005). Correlation between Mouse Potency and In Vitro Relative Potency for Human Papillomavirus Type 16 Virus-Like Particles and Gardasil® Vaccine Samples. Human Vaccines 1(5): 191-197. doi: 10.4161/hv.1.5.2126
  26. Sesardic D et al, (2003). Collaborative Study for the Validation of Serological Methods for Potency Testing of Diphtheria Toxoid Vaccines Extended Study: Correlation of Serology with in vivo Toxin Neutralisation. Link to article: https://www.edqm.eu/documents/52006/123862/bsp034-p1-ext-dserol.pdf
  27. Poirier B et al, (2000). In Vitro Potency Assay for Hepatitis A Vaccines: Development of a Unique Economical Test. Biologicals Volume 28, Issue 4, December 2000, Pages 247-256. 10.1006/biol.2001.0264

Safety testing

  1. Viviani L et al, (2022). Accelerating Global Deletion of the Abnormal Toxicity Test for vaccines and biologicals. Planning common next steps. A workshop Report. Biologicals 78: 17-26. doi: 10.1016/j.biologicals.2022.06.003
  2. Viviani L et al, (2020). Global harmonization of vaccine testing requirements: Making elimination of the ATT and TABST a concrete global achievement. Biologicals 63: 101-105. doi: 10.1016/j.biologicals.2019.10.007
  3. Wagner L et al, (2017). Towards replacement of the acellular pertussis vaccine safety test: Comparison of in vitro cytotoxic activity and in vivo activity in mice. Vaccine 35(51): 7160-7165. doi: 10.1016/j.vaccine.2017.10.082
  4. Arciniega J et al, (2016). Alternatives to HIST for acellular pertussis vaccines: progress and challenges in replacement. Pharmeur Bio Sci Notes 2015: 82–96. PMID: 27506225
  5. Dazhi J et al, (2016). Quantitative Detection of Vibrio cholera Toxin by Real-Time and Dynamic Cytotoxicity Monitoring. J Clin Microbiol 51(12) doi: 10.1128/jcm.01959-13
  6. Behrensdorf-Nicol H et al, (2015). "BINACLE" assay for in vitro detection of active tetanus neurotoxin in toxoids. ALTEX 32(2): 137-42. doi: 10.14573/altex.1412181
  7. Xing D et al, (2014). Whole-cell pertussis vaccine potency assays: the Kendrick test and alternative assays. Expert Review of Vaccines 13(10): 1175–1182. doi: 10.1586/14760584.2014.939636
  8. Isbrucker R et al, (2013). Transferability study of CHO cell clustering assays for monitoring of pertussis toxin activity in acellular pertussis vaccines. Pharmeur Bio Sci Notes. 2015: 97-114. PMID: 27506252
  9. Corbel M and Xing D (2004). Toxicity and potency evaluation of pertussis vaccines. Expert Rev Vaccines. 3(1): 89-101. doi: 10.1586/14760584.3.1.89
  10. McKenna A et al, (1995). Attenuated typhoid vaccine Salmonella typhi Ty21a: fingerprinting and quality control. Microbiology 141(8): 1993-2002. doi: 10.1099/13500872-141-8-1993
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