Lakiya Wimbish, Lonza, discusses the benefits of recombinant Factor C based endotoxin testing
Endotoxins are lipopolysaccharides found in the outer membrane of Gram-negative bacteria. These pyrogenic components can elicit an immune response if administered via the blood stream through contaminated parenteral drugs or other medical devices. Hence, bacterial endotoxin testing (BET) needs be carried out for all such products in order to prevent any adverse effects.
In the 1950s, Fred Bang discovered that horseshoe crab (Limulus polyphemus, Tachypleus gigas and T. tridentatus) blood reacts in the presence of endotoxin. This is due to the presence of Factor C, a component that activates the clotting cascade (Figure 1) and forms the basis of the Limulus Amebocyte Lysate (LAL) and Tachypleus Amebocyte Lysate (TAL) assays used in BET today.
Figure 1: Comparing LAL turbidimetric and chromogenic assays to fluorescence-based Pyrogene recombinant Factor C assay.
The industry is heavily reliant upon the supply of LAL/TAL for endotoxin testing. However, scientists have now developed a recombinant version of Factor C (rFC) that is comparable to the natural equivalent. One of the reasons the industry has perhaps been reluctant to adopt this alternative is the fact that the pharmacopoeial regulations currently necessitate an additional validation procedure to be carried out. However, this is not as complicated or time-consuming as it may appear.
The need for alternative tests
The need for the widespread adoption of an alternative assay is becoming increasingly important due the fact that the demand for LAL/TAL may soon exceed the supply. This is due to an increasing number of new biologics being developed, the growing impact of personalised medicine and the maturing Asia-Pacific markets. In addition, as with any natural resource, crab populations are at risk of mismanagement and loss. Therefore, adopting rFC-based methods would not only meet European and global initiatives to reduce animal use in testing, but would also decrease the demand made on the crabs, a finite resource.
In addition to negating the need for an animal source, rFC products, such as Lonza’s fluorescence-based Pyrogene rFC assay, perform comparably or better than LAL/TAL-based methods for some applications, and offer a number of additional benefits to the user. These include improved lot-to-lot consistency, enhanced endotoxin specificity, statistically more robust spike recovery, ease-of-use and a suitable sensitivity range (0.005 EU/ml – 5 EU/ml).
Validating the alternative
So what is holding back manufacturers from adopting rFC-based assays? According to the United States Pharmacopeia (USP) rFC-based assays are listed as ‘alternative methods’, and must be validated as per USP <1225> or International Conference on Harmonisation (ICH) Q2B. This means that their use requires extended validation over and above the compendial methods, to prove the alternative tests meet the intended analytical application. While these steps do require some additional effort, these can be accomplished in one to three days of following a well-structured protocol, alleviating any concerns of significantly lengthening the testing workflow.
The procedure includes four steps. The initial qualification involves an assessment of the reagents and testing equipment to establish whether the current system is functioning properly. The second step determines the level of dilution required to overcome product inhibition or enhancement. Thirdly, the alternative method must be validated and should show that the rFC is capable of achieving comparable results to the current method. In order for the regulators to evaluate the validity of the proposed method, submissions should contain the following: 1) a rationale for using the test, such as the fact that it reduces a reliance on animals and offers enhanced endotoxin specificity and reproducibility; 2) a complete description of the proposed analytical procedure; and 3) data elements that demonstrate the analytical performance of the procedure. The latter should include the following parameters: accuracy, precision, specificity, linearity, range, detection limit, quantitation limit, and robustness. The fourth and final step of the validation process requires a product-specific validation to ensure it is possible to generate consistent results using the chosen method.
Conclusion
Clearly, there is a need for LAL/TAL assay alternatives due to the growing demand on resources. Fortunately, rFC-based methods are available that offer a number of benefits to the user. Although defined as an alternative by the pharmacopoeia, these tests are simply a fluorescence-based version of the existing LAL/TAL assay and do not require much additional effort to implement. Hence, companies should not be dissuaded from adopting these methods. To make the switch even easier, companies such as Lonza have compiled pre-developed documentation to assist with regulatory submissions and can also help users to incorporate the technology in their testing workflow. It seems likely that rFC will become the go-to solution for BET in the future, and many forward-thinking manufacturers are already adopting it into their processes.