How did Lonza’s development and commercialization of the first FDA licensed endpoint quantitative LAL assay in the 1980s impact the industry?
The development of the endpoint chromogenic technology was a critical step in the development of the kinetic chromogenic assays that are in regular use today around the world. The discovery was based on determining the sequence of amino acids at the cleavage site of coagulin, the last step in the enzymatic cascade leading to gel clot formulation in the classical gel-clot LAL assay. Once this sequence was known, a peptide could be sequenced that could be attached to a chromophore. When the chromophore was cleaved from the peptide, a yellow color was generated. The amount of color generated by enzymatic cleavage was proportionate to the amount of endotoxin in the sample, and could be measured using a spectrophotometer. Generation of color development, rather than a clot or turbidity, made it easier to test samples that were not colorless.
Improvements in the technology lead to measuring the time for the color development to cross a specific OD threshold, which was related to the endotoxin content of a sample. That onset time could be interpolated into a standard curve of endotoxin content of several logs. This was the Kinetic Chromogenic LAL assay that is so familiar today. This technology also increased sensitivity to overcome the assay interferences of the product.
What are the benefits of Lonza’s PyroGene™ assay?
PyroGene™ is a recombinant form of the first enzyme in the classical LAL cascade (Factor C) found in horseshoe crabs. When Factor C encounters endotoxin, there is a conformational change which causes the cleavage of the next enzyme in the LAL cascade. In the case of PyroGene™, Factor C cleaves a fluorogenic substrate that will cause luminescence instead. The advantage of using a recombinant form of Factor C coupled to a fluorogenic substrate is that it removes the remainder of the enzyme cascade, and reduces the potential for assay interferences. Lot-to-lot variability is also controlled. It also eliminates the use of live animals needed to produce LAL, and replaces them with an enzyme that can be produced any time of the year. Therefore production of the enzyme is no longer reliant on the availability of horseshoe crabs in the summer months. PyroGene™ does not contain another enzyme found in the horseshoe crab’s primitive immune system, Factor G, which reacts to glucans, and causes false positive reactions in the classical LAL assay.
Discuss the importance of endotoxin detection software and Lonza’s investments in this area.
Software reliability is a key prerequisite in a kinetic assay. Lonza’s WinKQCL™ software is unsurpassed in reliability and ease of use, as well as being fully compliant with 21 CFR Part 11. This software provides the analyst with the ability to set up an assay in a plate reader and walk away, knowing that the assay performance will be monitored and reported accurately.
What is your reaction to the FDA’s latest guidance on pyrogen and endotoxin testing?
The revision to the original 1987 Guidance was long overdue, as it no longer reflected FDA’s current thinking, and was obsolete. FDA also stated that USP and AAMI documents contained adequate information for assay performance.
One positive aspect is that FDA specifically said that Alternative Test Methods, such as recombinant Factor C assays (PyroGene™) are acceptable for product release, when properly validated. Therefore, customers should feel comfortable using PyroGene™ for final product release, and in regulatory submissions.