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Published on: June 24, 2019
Endotoxin testing of proteins for parenteral administration using the Mono Mac 6 assay
L Moesby1, E W Hansen, J D Christensen
1Department of Pharmacology, The Royal Danish School of Pharmacy, 2 Universitetsparken, 2100 Copenhagen, Denmark. LM@mail.dfh.dk
Background:
Pharmaceutical products containing proteins cause problems in testing for endotoxin and pyrogens. Many proteins interfere with the LAL test and the proteins are immunogenic in rabbits. The monocytic cell line Mono Mac 6 is an alternative assay for detection of endotoxin and other pyrogens.
Objective:
To evaluate the use of the Mono Mac 6 assay for quantitative detection of endotoxin in proteins.
Method:
The quantitative detection of endotoxin in the three pharmaceutical products human albumin, gamma-globulin and somatropin was evaluated.
Results:
For the three proteins the detection limit of the Mono Mac 6 assay was far below the threshold endotoxin limit described by the European Pharmacopoeia. Interference of two of the proteins with the Mono Mac 6 assay was observed, but the problems could be overcome either by dilution of the product or by comparison of the test with an endotoxin standard curve prepared in a solution of the respective pyrogen-free protein.
Conclusion:
The Mono Mac 6 assay is a reliable method for quantitative detection of endotoxin in proteins.
Insights
The Mono Mac 6 cell line assay reliably quantifies endotoxin in protein-based pharmaceuticals. This method overcomes interference issues, offering a dependable alternative for pyrogen testing in sensitive protein products.
Area of Science:
- Biotechnology
- Pharmaceutical Analysis
- Cell-based Assays
Background:
- Protein-based pharmaceuticals pose challenges for traditional endotoxin and pyrogen testing.
- The Limulus Amebocyte Lysate (LAL) test is often interfered with by proteins.
- Rabbit immunogenicity tests present ethical and practical limitations.
Purpose of the Study:
- To assess the efficacy of the Mono Mac 6 monocytic cell line assay for endotoxin detection in protein pharmaceuticals.
- To evaluate the quantitative accuracy of the Mono Mac 6 assay for endotoxin levels in specific protein products.
Main Methods:
- Quantitative evaluation of endotoxin detection in human albumin, gamma-globulin, and somatropin using the Mono Mac 6 assay.
- Investigating and addressing protein interference with the assay.
Main Results:
- The Mono Mac 6 assay demonstrated a detection limit well below European Pharmacopoeia thresholds for all tested proteins.
- Interference from two protein products was observed but successfully managed through product dilution or using protein-specific standard curves.
- The assay proved effective for quantitative endotoxin determination in the presence of proteinaceous substances.
Conclusions:
- The Mono Mac 6 assay is a robust and reliable method for the quantitative detection of endotoxin in protein-containing pharmaceutical products.
- This cell-based assay offers a viable alternative to conventional pyrogen testing methods for complex biological drugs.
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