It is currently a very interesting and dynamic time for the endotoxin testing community, as highlighted at Lonza’s recent Global Endotoxin Testing Summit during which vendors, regulatory bodies, pharmaceutical manufacturers, and conservationists came together to share their thoughts and perspectives on some of the industry’s key issues. Some of the challenges being addressed at the summit and discussed in detail here include methods to overcome the issue of low endotoxin recovery (LER), the need for alternative tests in order to safeguard lysate supply and conserve the horseshoe crab, a guide to validating these alternative assays and the move toward automated systems.
OVERCOMING LOW ENDOTOXIN RECOVERY
LER is widely regarded as a real challenge for the endotoxin testing community and a problem that must be overcome. As Ingo Spreitzer, deputy head of the Microbial Safety Department of the Paul-Ehrlich-Institute (PEI) and European Directorate for the Quality of Medicines and Healthcare (EDQM), whose group published the new European Pharmacopoeial (EP) Chapter 5.1.10, Bacterial Endotoxins Ph. Eur. Policy for Substances for Pharmaceutical Use, proposed, “LER is the most important issue in endotoxin testing today because it is affecting the testing of current products that are already on the market.”
LER occurs when various combinations of excipients used in biopharmaceutical production mask known amounts of endotoxin in undiluted samples. This can result in false negatives if added endotoxin is not adequately recovered and ultimately challenges the validity of these results.
While regulators, pharmaceutical manufacturers and test vendors are aware of the issue, there is still much debate concerning the mechanisms behind LER and how endotoxin masking might be overcome. Allen Burgenson, U.S. Regulatory Affairs manager at Lonza, thinks, “LER is a new type of inhibition. We’ve seen inhibition before and we solved it. There are a number of methods by which LER can be overcome before you have to go into the chemistry of demasking. The issue of LER is manageable, and while I think it is an important technical problem, we do not necessarily have a public health problem.”
Alan Baines, UK Strategic Projects head at Lonza, suggested that, “…when considering new biological license applications, manufacturers should be aware of the potential issues with LER when using polysorbate in combination with citrate or phosphate buffers. Finding the right combination could side-step potential problems. For existing products, it is likely that additional sample treatment steps will be needed to overcome the masking effect, as changes to formulations are usually a much less attractive option.”
To overcome the masking effect, Johannes Reich, PhD student at the University of Regensburg, has been working in conjunction with Hyglos GmbH to gain a better understanding of the aggregation and interaction of lipopolysaccharides in endotoxin testing. His theory assumes that LER is the result of endotoxin aggregate breakdown into monomers, which are subsequently embedded in surfactant micelles. He proposes that this process is reversible and as such, endotoxin demasking will require adjustments in magnesium and calcium, pH and/or the addition of polyanionic dispersants such as Pyrosperse, in order to reassemble the aggregates to allow for sufficient endotoxin recovery.
However, demasking is still an active area of contention among some industry members as Kevin Williams, senior scientist for Endotoxin Detection at Lonza, believes, “…many people are resisting using the demasking protocol as they want to maintain the simplicity of the test. I can certainly understand. However, you can’t maintain simplicity by denying that complexity exists. So what we hope to do is to inform and educate at least the manufacturers of biologics such as monoclonal antibodies about the importance of addressing LER, as these are life-saving drugs and it’s a significant and growing market area.”
This reluctance is understandable, as carrying out an investigation into LER could delay the release of new products and cause significant financial issues if a product cannot be released within a reasonable timeframe. This is a particular problem for biological products, as they often have a very short shelf-life and require a fast turnaround time.
Regulators can also add additional time pressures; if a product is tested and shown to display LER, manufacturers will need to overcome the issue within a given timeframe, as stipulated by the FDA (typically one year but this can vary). The manufacturing delays introduced by the need to overcome the issue will likely be significant. In addition, biological products for which no defined hold-time has been established will need to be tested as soon as possible. This may cause a back-up in QC testing, as products that exhibit the signs of early or somewhat instantaneous LER will need to be pushed ahead of other products where a longer hold-time has been established. If no clear hold-time can be established during the development stage due to LER, significant time and money may need to be invested to uncover better formulations that exclude the excipients known to be associated with LER.