The bacterial endotoxin test is a mainstay of quality and safety in pharmaceutical manufacturing. However, the need for improved efficiency and standardisation across the industry as a whole is driving considerable discussion regarding the best use of the limulus amebocyte lysate (LAL) test to maximise quality and patient safety.
Here, we review some of the hot topics that dominate the agenda when experts from across the endotoxin community are brought together, as demonstrated at the 2nd Global Endotoxin Summit.
Such topics include the considerable debate about the best way to deal with low endotoxin recovery (LER), concerns regarding the appropriate testing of pyrogenic contamination in pharmaceutical products, and disagreement about the most effective standards to use for spike-in controls. Data integrity is also growing in importance.
Patient safety
Endotoxin testing is a crucial aspect of manufacturing parenteral pharmaceuticals and medical devices. Without it, there would be considerable risk to patient safety. Endotoxins form part of the outer membrane of Gram-negative bacteria. They are a type of pyrogen; that is, they induce fever if introduced into the bloodstream. To protect patients, all parenteral pharmaceutical products labelled as sterile or pyrogen-free must be tested to ensure they are negative for endotoxins.
There are two key methods used for pyrogen testing. The rabbit pyrogen test (RPT) involves injecting a small amount of the test sample into a rabbit’s bloodstream and watching for a temperature rise. This test is required by law in the US for biological products, but can be waived if an alternative method is shown to be equivalent. A widely accepted substitute is a bacterial endotoxin test based on LAL. In the presence of an endotoxin, an enzyme cascade results in the formation of a blood clot, which envelopes the invading endotoxin. This assay is accurate and reliable and has almost replaced the RPT for endotoxin testing.
Low endotoxin recovery
However, one challenge affecting the LAL test is the phenomenon of LER, which continues to cause debate among the endotoxin testing community. LER is defined as the failure to detect a known amount of spiked endotoxin in an undiluted product during multiple time points. It is thought to be attributable to particular excipients and differs from the inhibition or interference of endotoxin tests caused by pH, high divalent ion concentrations, chelators, serine proteases and beta-glucans, which can usually be overcome using a simple pretreatment such as dilution.
The top question for many endotoxin experts is whether LER actually poses a risk to patient safety. There have been no recorded incidences of pyrogenicity caused by a product that had previously tested negative using the LAL test. However, LAL actually detects endotoxin contamination, rather than being a direct measure of overall pyrogenic potential. This is a concern for some who believe that it would be more important to test for the fever-inducing activity of endotoxin.
Regardless of these ongoing discussions, pharmaceutical manufacturers must continue to test for LER while regulatory bodies, particularly the US FDA, continue to demand evidence that LER is not an issue for susceptible products. Meanwhile, research continues into the causes and mechanisms of LER in an effort to identify the exact conditions that trigger its occurrence. Unfortunately, the more research data that is generated, the more complex the phenomenon seems to become.
One such complexity is the different masking susceptibilities of endotoxins from different sources (such as naturally occurring endotoxin versus purified, and therefore artificial, reference standards). As these alterative endotoxins can react differently, there is unlikely to be one all-encompassing solution to LER.
Search for a standard
Currently, there are no standardised principles and procedures for performing spike/hold-time studies to determine whether LER has occurred. This is particularly relevant as sample preparation procedures can greatly influence the ability to recover endotoxin. In addition, there is no consensus on what type of endotoxin is the most relevant source to use in spike/hold studies. Three potential sources are
Reference Standard Endotoxin (RSE): Lipopolysaccharide (LPS) is just one portion the endotoxin molecule. RSE contains LPS that has been extracted from E. coli 0113:H10 and purified. As such, it doesn’t exist in nature.
Control Standard Endotoxin (CSE): These are endotoxin preparations other than international or national reference standards that are traceable in their calibration to the RSE. CSEs are manufactured and certified by an LAL reagent manufacturer for use with a specific lot of reagent under defined assay conditions. This provides a more economical alternative to RSE.
Naturally Occurring Endotoxin (NOE): Endotoxins are constituents of the outer membrane of Gram-negative bacteria. The natural endotoxin complex contains many bacterial cell wall components including phospholipids, lipoproteins and lipopolysaccharide, which is the biologically active component. NOEs are not purified so they can lead to varying results.
Some experts feel that NOE most accurately reflects a contamination event and therefore might be a more relevant analyte for hold-time studies than purified endotoxins. The theory is that NOEs are recoverable under typical LER conditions without the need for demasking, and it’s possible that the presence of protein and other cell debris in NOEs makes the lipopolysaccharide more robust. However, variability in results is a problem, and without a standardised process for producing NOE, it can’t be proven whether NOEs provide a useful alternative to purified lipopolysaccharides.
Not only are RSE, CSE and NOE all different, but natural LPS molecules also vary depending on their source. The basic LPS structure is conserved but the lipid length and the number of acyl groups may differ between different species, which can result in highly variable pyrogenicity. There are still a lot of questions surrounding LER and further research is needed. Discussions around the causes of LER and the true purpose of the LAL test (pyrogen or contaminant detector) have prompted further questions about which standard should be used for all endotoxin testing. All these topics are interlinked and answers are needed before rational, data-driven decisions can be made.
Data integrity
In recent years, there has been an increased focus on how pharmaceutical QC data is collected, managed and stored. In this digital age, a surprising number of companies are still relying on paper-based environmental monitoring approaches. This obviously carries a significant risk of human error. It also takes a lot of time to collect and interpret information captured on paper. Newer paperless systems can improve the reliability of this information and seamlessly integrate with laboratory information management systems (LIMS) to link QC and product manufacture.
The regulatory requirements for data integrity — the overall completeness, accuracy and consistency of data — are not new and apply to both paper and electronic data. However, it’s fair to say that as more and more pharmaceutical processes become automated, data integrity has become a renewed focus for FDA. As a result, GMP inspectors are receiving extensive training in the subject and have high expectations. As well as conducting data integrity specific inspections at high-risk sites, FDA has published new data integrity guidelines.
There are a number of advantages related to adopting a paperless approach to data collection and processing. It is more accurate, efficient and cost-effective. It also provides the opportunity to draw together separate pieces of information, thereby improving data tracking, analysis and access. From a practical point of view, paper-based systems are inefficient and not suited to pharmaceutical manufacturing environments — as all materials, including paper, must be sterilised before entering the cleanroom.
To ensure regulatory compliance, paperless systems must ensure data quality and integrity. The design of these systems (including software, hardware, personnel and documentation) must include controls that validate a system for its intended use. FDA made data audit trails mandatory, so the system should make stored data as easy to locate and view as it was to input the data in the first place.
In addition to having a reliable, efficient system, data integrity relies on an organisation adopting an appropriate ethos. This must encourage the open reporting of errors, provide appropriate training and raise awareness of data integrity across all relevant areas of the organisation. With this in place, data integrity offers the opportunity to improve quality across the manufacturing process.
In the US, a number of initiatives are in place to ensure that horseshoe crab numbers are maintained
Protecting them, protecting us
For decades, we have relied on the blood of the horseshoe crab to protect us by ensuring that our pharmaceuticals and medical devices are free from bacterial endotoxins. For the purpose of endotoxin testing, the industry collects the blue blood of two species of horseshoe crab — the North Atlantic Limulus polyphemus, which is used for LAL tests; and the Asian Tachypleus tridentatus, for TAL assays. In the US, a number of initiatives are in place to ensure that horseshoe crab numbers are maintained, and these have mostly proven successful. In Asia, the situation is more complex as the natural habitat spans several countries with different needs, priorities and politics.
Globally, a key threat to the horseshoe crab is its use as bait by the conch and eel fisheries. This is carefully managed in the US to ensure sustainability, and the population is stable throughout much of the geographic range; however, it continues to decline in the New York and New England states, which may prompt tighter restrictions. In addition to concerns about population, demand for LAL and TAL continues to grow as global demand for pharmaceuticals rises. This includes increasing global vaccine production and the burgeoning needs of emerging markets.
As such, pharmaceutical companies are being encouraged to support conservation efforts. In recent years, contributions from Lonza have enabled the Ecological Research & Development Group (ERDG) to expand two online resources — the teachers’ toolbox and the research database — and to fund an investigation project researching alternative bait to horseshoe crabs. The research database is a repository for any data or papers that relate to LAL/TAL and horseshoe crabs. Researchers and experts from across the industry are being asked to upload material.
The pharmaceutical industry itself is well placed to help conserve the horseshoe crab. As the purchasers of endotoxin detection products, it is important to make responsible decisions in terms of supply line ethics to ensure sustainability and conservation of the horseshoe crab.
Conclusion
Demand for endotoxin testing continues to grow, along with a pressure to further improve efficiency and quality. As part of this, we are seeing an increased focus on process optimisation, automation and data integrity. At the same time, LER continues to cause issues for pharmaceutical biologics manufacturers and raises many unresolved questions about the source of endotoxin and its effect on the LAL test, making it hard for the industry to standardise on a particular method and process. Further research is needed before the mechanisms and conditions causing this phenomenon can be resolved.
Perhaps more fundamentally, there is still disagreement among the industry about whether LER poses a risk to patient safety and whether it’s more important to detect endotoxin contamination or to measure overall pyrogenicity. This is a very interesting time for the endotoxin testing community and the research and debates are likely to continue for some time yet.
