Speaking to Katrin Pauls from Lonza about the launch of the company's PyroCell MAT, which uses human serum instead of foetal bovine serum for pharmaceutical testing, and how it changes the status quo
Q: Could you explain the differences between the new PyroCell MAT, the foetal bovine serum one, and the rabbit pyrogen test?
A: The Rabbit Pyrogen Test (RPT) and the Monocyte Activation Test (MAT) are used to detect pyrogens (endotoxins and non-endotoxin pyrogens). The main difference is the RPT measures a fever response in rabbits and, therefore, does not provide an experimental control. The MAT is a cell-based assay that measures inflammatory cytokines which indicate a fever response. MAT was developed to become a replacement for the RPT.
Lonza offers two PyroCell MAT Kits: The PyroCell MAT System utilises foetal bovine serum (FBS) in the media used to grow cells, and shows higher sensitivity towards endotoxins. The PyroCell MAT HS System issues human serum instead of FBS, and is more sensitive toward non-endotoxin pyrogens (NEP).
Pyrogens are fever-inducing substances primarily derived from microorganisms or chemical substances
Q: What are the functional differences between an endotoxin and a non-endotoxin pyrogen? How do their impacts on humans differ? Do you have some examples of types of organisms that cause each?
A: Pyrogens are fever-inducing substances primarily derived from microorganisms or chemical substances, e.g. primary packaging materials. Pyrogens entering the bloodstream may interact with the host immune system to cause inflammation, fever, chills, shock and, in severe cases, death. Pyrogens that derive from causes other than gram-negative bacteria are collectively referred to as non-endotoxin pyrogens (NEPs).
Endotoxins are the most prevalent type of pyrogen and are derived from the cell wall of gram-negative bacteria such as E.coli, Enterobacteriaceae, Pseudomonas, Salmonella, Shigella, etc.
Non-endotoxin pyrogens derive from gram-positive bacteria (such as bacilli, and cocci), Yeast or Moulds, glycans from filter materials, virus, DNA/ RNA, and other substances.
Q: Why is there a need for enhanced sensitivity in non-endotoxin pyrogen testing?
A: Many non-endotoxin pyrogens may not be life threatening for healthy patients but become dangerous for immune-suppressed patients. Since NEP’s can also be derived from organic materials, early detection is helpful to ensure manufacturing lines are in control. When manufacturing of a product is established, manufacturers do a risk analysis to determine where and what type of contamination may be introduced.
Q: Is there much of a financial difference between the old animal test and the new one?
A: There is a wide regional difference driven by laws and regulations in the pricing of RPT. The main benefit of the MAT is time. In Europe, experimental animal housing needs an exceptional license, another license is needed for each test. Fresh rabbits are trained for 55 days on average. Once established in the lab, the MAT is a 2-day test.
Q: How long does the cell culture step take? How long does the entire test take on average?
A: The cell culture step takes 18-24 Hours. The entire test takes 2 days with about 6-8 hours of handling time
Q: Why was bovine the normal serum used prior to this?
A: Bovine serum is the most widely used serum-supplement for in vitro cell culture of eukaryotic cells.
Q: What is the biggest challenge with regard to manufacturing the human serum?
A: There is no real challenge for manufacturing. Human serum is collected from voluntary blood donation, with documented informed consent, and no harm to the human. The main benefit of using human serum is decreasing the reliance on animal products, such as the foetal bovine serum.
Q: What should a pharmaceutical company do if they have a positive test?
A: These specific procedures would be defined by the customers quality management system. In general, upon confirmation of the positive result, an investigation would be initiated to determine root cause and a course for preventative/corrective actions taken for the future. The contaminated batch of material would be discarded.
Q: How should pharmaceutical companies integrate the MAT test into this contamination testing?
A: Testing is done at critical points during the manufacturing process. For new products, MAT is employed as the product transitions to clinical development.
For routine products, MAT is employed when a risk for NEP exists, and whenever the traditional bacterial endotoxins tests are not applicable.
Q: It is great to be decreasing reliance on animal testing, is there any hesitancy to use new solutions that you have seen?
A: There is some resistance to new products because MAT is not harmonised across pharmacopoeia making it difficult to produce one data set for global distribution. MAT is gaining prevalence as the pharmaceutical industry requires robust tests suitable for use with the diversity of emerging biologics products and reduces the use of experimental animals. Ph. Eur. has put an end date on the RPT in 2026.