In the pharmaceutical industry, customers dealing with challenging products, particularly those in the cell and gene therapy sector, confront unique hurdles when it comes to ensuring product safety. This article explores the unique challenges faced by these customers in endotoxin testing and introduces our innovative solutions to address these challenges.

For customers working with challenging samples, particularly those in cell and gene therapy, we have engineered ENDOLISA. It is a black 96-well plate coated with bacteriophage recombinant proteins specific (in nature) to Gram-negative bacteria.  Paired with a set of reagents (rFC, wash buffer, and binding buffer), it functions much like an ELISA test, effectively binding and purging interfering substances, enabling highly sensitive testing even at low dilutions. 

Figure 1 below demonstrates ENDOLISA’s exceptional ability to overcome interferences in common solutions, such as buffer/pH, salt, chaotropic, solvents, detergents, chelators, protease inhibitors, and antibiotics.  It showcases substantial improvements compared to conventional LAL testing, with some cases achieving up to 1000X sensitivity, notably with Benzamidine.  

For customers working with challenging samples, particularly those in cell and gene therapy, we have engineered ENDOLISA. It is a black 96-well plate coated with bacteriophage recombinant proteins specific (in nature) to Gram-negative bacteria.  Paired with a set of reagents (rFC, wash buffer, and binding buffer), it functions much like an ELISA test, effectively binding and purging interfering substances, enabling highly sensitive testing even at low dilutions. 

Figure 1 below demonstrates ENDOLISA’s exceptional ability to overcome interferences in common solutions, such as buffer/pH, salt, chaotropic, solvents, detergents, chelators, protease inhibitors, and antibiotics.  It showcases substantial improvements compared to conventional LAL testing, with some cases achieving up to 1000X sensitivity, notably with Benzamidine.  

Low Endotoxin Recovery (LER) represents a significant concern, particularly for customers dealing with complex products. LER manifests when endotoxins are present but remain undetectable due to specific formulation conditions. Chen and Vinther’s original claims (2013) that endotoxin spiked into some common test matrix types (chelator and surfactant) could not be subsequently detected have been confirmed with many subsequent tests using many different substances.¹

Regulatory agencies including the FDA now mandate hold time studies (HTS) during the Biologic License Application (BLA) submission to ensure endotoxin detection in matrices that include a chelator, such as phosphate or citrate buffers or EDTA, and a surfactant, such as polysorbate.

The Parenteral Drug Association (PDA) offers valuable insights into combating LER through Technical Report No. 82 (TR82). This report underscores the importance of HTS in identifying conditions that give rise to LER, rendering contaminants difficult to detect. Within TR82, one solution highlighted is the bioMérieux product, Endo-RS®, which adeptly addresses LER challenges.

ENDO-RS® stands as a comprehensive kit meticulously devised to demask endotoxins that often evade detection in Factor C-based tests.  To achieve an optimal demasking result, concentrations of components of the ENDO-RS kit (A through E) as well as of further components (as needed) are adjusted in volume (concentration used) to match the particular conditions of the masked endotoxin. 

ENDO-RS® effectively restores samples to their original state before endotoxin disaggregation. This step is pivotal in re-establishing micelle formation, especially in the presence of chelators and surfactants. 

After successfully navigating the initial development phase, which may involve some complexity depending on the masking intensity, the subsequent application of the final test, utilizing specific components (A-E) for product testing, becomes a straightforward process. This ensures a user-friendly experience for our website customers.

Let's examine a real-world illustration. When testing samples related to cell and gene therapy or similarly intricate products, ENDO-RS® emerges as an invaluable tool in revealing a starting point for further development. By applying components, A-E from the kit, customers can fine-tune concentration and mixing variables to attain consistent recovery. The masked samples that have been held for 7 days, typically at room and refrigeration temperatures, are tested using the ENDO-RS treatment. An example of initial recovery is shown below for a CAR-T type sample.  

From the starting points found above (in green), one can adjust the concentration and mixing variables to achieve better and more consistent routine recovery to achieve a finalized test that no longer requires such effort in the development and becomes a test treatment like any other Limulus-based test treatment.

In conclusion, the journey of endotoxin testing for customers working with complex and difficult products like in the cell and gene therapy industry presents formidable challenges. As customers in this field, you face unique challenges, but with solutions like ENDOLISA and ENDO-RS® the path to reliable endotoxin detection becomes more attainable. Our commitment is to make your journey towards product safety as smooth as possible, ensuring that your therapies are not only groundbreaking but also safe for patients.