Comparison of pyrogen assays by testing products exhibiting low endotoxin recovery

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Tammy L. Thurman
Carol J. Lahti
Jeanne M. Mateffy
Ren-Yo Forng
Friedrich von Wintzingerode
Lindsey M. Silva
Sven M. Deutschmann
Ned M. Mozier

Abstract

The use of pyrogen tests to assess the risk of endotoxin in biological products has increased recently due to concerns of some regulatory authorities about products exhibiting low endotoxin recovery (LER). Manufacturers increasingly seek to reduce the use of animals unless essential to assure patient safety. The current study compares the ability of the monocyte activation test (MAT) and the bacterial endotoxin test (BET) to the rabbit pyrogen test (RPT) to detect endotoxin spikes in samples of products shown to exhibit LER. Product samples or water were spiked with endotoxin and held for three days or tested immediately in the BET, the RPT and two variations of the MAT at the same time. Results show high sensitivity to endotoxin of both the BET and MAT and much lower sensitivity in the RPT, indicating that much higher levels of reference standard endotoxin are required to induce pyrogenicity in the RPT than the 5 endotoxin units (EU) per kg common threshold. The results of the BET and MAT correlated well for the detection of endotoxin spike in water. We also show that LER (masking of endotoxin) found in the BET is also seen in the MAT and RPT, suggesting that the products themselves elicit a biological inactivation of spiked endotoxin over time, thereby rendering it less or non-pyrogenic. We conclude the non-animal MAT option is a suitable replacement for the RPT to measure spiked endotoxin in biopharmaceuticals.

Article Details

How to Cite
Thurman, T. L., Lahti, C. J., Mateffy, J. M., Forng, R.-Y., von Wintzingerode, F., Silva, L. M., Deutschmann, S. M. and Mozier, N. M. (2022) “Comparison of pyrogen assays by testing products exhibiting low endotoxin recovery”, ALTEX - Alternatives to animal experimentation. doi: 10.14573/altex.2202021.
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References

Bolden, J., Knutsen, C., Levin, J. 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. 74, 602-611. doi:10.5731/pdajpst.2020.012187

Bleeker, W.K., de Groot, E.M., den Boer, P.J. et al. (1994). Measurement of interleukin-6 production by monocytes for in vitro safety testing of hemoglobin solutions. Artif. Cells Blood Substit. Immobil. Biotechnol. 22, 835-840. doi:10.3109/10731199409117918

Chen, D., von Wintzingerode, F., Barlasov-Brown, J. et al. (2019). Low Endotoxin Recovery. Parenteral Drug Association© Technical Report No. 82, 1-128. ISBN # 9781945584077 https://techpubportal.pda.org

EDQM – European Directorate for the Quality of Medicines and HealthCare (2020a). Chapter 2.6.8 Pyrogens. European Pharmacopoeia. https://pheur.edqm.eu/home

EDQM (2020b). Chapter 2.6.14 Bacterial Endotoxins. European Pharmacopoeia. https://pheur.edqm.eu/home

EDQM (2020c). Chapter 2.6.30 Monocyte Activation Test. European Pharmacopoeia. https://pheur.edqm.eu/home

EDQM (2020d). Chapter 5.1.10 Guidelines for Using the Test for Bacterial Endotoxins. European Pharmacopoeia. https://pheur.edqm.eu/home

EDQM (2021a). Chapter 2.6.32 Test for bacterial endotoxins using recombinant factor C (rFc). European Pharmacopoeia. https://pheur.edqm.eu/home

EDQM (2021b). European Pharmacopoeia to put an end to the rabbit pyrogen test. European Pharmacopoeia Animal Testing News. Strasbourg, France. https://www.edqm.eu/en/news/european-pharmacopoeia-put-end-rabbit-pyrogen-test (Accessed 23 May 2022).

Fennrich, S., Fischer, M., Hartung, T. et al. (1999). Detection of endotoxins and other pyrogens using human whole blood. In Sesardic, D., Brown, F., Hendriksen, C.F.M. (eds), Developments in Biological Standardization 101, 131–139.

Hartung, T. (2021). Pyrogen testing revisited on the occasion of the 25th anniversary of the whole blood monocyte activation test. ALTEX 38, 3-19. doi:10.14573/altex.2101051

Helle, M., Brakenhoff, J.P., De Groot, E.R. et al. (1988). Interleukin 6 is involved in interleukin 1-induced activities. Eur. J. Immunol. 18, 957-959. doi:10.1002/eji.1830180619

Hoffman, S., Luderitz-Puchel, U., Montag, T. et al. (2005). Optimisation of Pyrogen Testing in Parenterals According to Different Pharmacopoeias by Probabilistic Modelling. Journal of Endotoxin Research. 11, 25-31. doi:10.1179/096805105225006678

Kikkert, R., Bulder, I., de Groot, E.R. et al. (2007). Potentiation of Toll-like receptor-induced cytokine production by (1-->3)-beta-D-glucans: implications for the monocyte activation test. J. Endotoxin Res. 13, 140-149. doi:10.1177/0968051907080024

Kikuchi, H., Hashima, Y., Fukui, C. et al. (2017). Collaborative Study on the Bacterial Endotoxins Test Using Recombinant Factor C-Based Procedure for Detection of Lipopolysaccharides. Pharm. Med. Device Regul. Sci. 48, 252–260.

Molenaar-de Backer, M. W. A., Gitz, E., Dieker, M. et al. (2021) Performance of monocyte activation test supplemented with human serum compared to fetal bovine serum. ALTEX 38, 307–315. doi:10.14573/altex.2008261

Mozier, N. (2019). Chapter 13: Monocyte Activation Test as a Developmental Tool for Pyrogens. In Kevin L. Williams (ed.), Endotoxin Detection and Control in Pharma, Limulus and Mammalian Systems (537-545). Springer Nature Switzerland AG. ISBN: 978-3-030-17148-3. doi:10.1007/978-3-030-17148-3_15

Solati, S., Aarden, L., Zeerleder, S. and Wouters, D. (2015) An improved monocyte activation test using cryopreserved pooled human mononuclear cells. Innate Immun. 21, 677-684. doi:10.1177/1753425915583365

Tindall, B., Demircioglu, D., and Uhlig, T. (2021). Recombinant bacterial endotoxin testing: a proven solution. BioTechniques 70, 290-300. doi:10.2144/btn-2020-0165

US FDA (2012) Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/guidance-industry-pyrogen-and-endotoxins-testing-questions-and-answers (Accessed: 23 May 2022).

USP – United States Pharmacopeia. General Chapter <85>. Bacterial Endotoxins Test. doi:10.31003/USPNF_M98830_02_01

USP. General Chapter <151> Pyrogen Test. doi:10.31003/USPNF_M98900_01_01