Endotoxin testing is an essential part of the manufacturing process for parenteral products. Without this critical step, patient health and safety would be compromised.
Endotoxins are derivatives of bacterial cell membranes that can induce unfavourable symptoms in patients, including fever, when not passed through the digestive system. Current endotoxin tests employ assays using lysate of one of two horseshoe crab genera: Limulus or Tachypleus. Limulus amoebocyte lysate (LAL) and Tachypleus amoebocyte lysate (TAL) coagulate when in contact with endotoxins and this highly sensitive reaction forms the basis for the assays. While this test currently remains a key resource for the pharmaceutical industry, challenges are emerging, and these challenges are being met by innovative solutions that are pushing the endotoxin testing community into new directions.
As the global population grows and developing nations become wealthier, healthcare demands increase, including the demand for parenteral medicines. These medicines all need to be tested for endotoxins and will therefore enhance the demand for LAL and TAL, the former being the sole lysate used in Western nations and the latter being used predominantly in Asia. This will place great pressure on the source of these lysates: the blue blood of horseshoe crabs.
Regulations such as state-by-state fishing quotas have been put in place in the US to protect the North American L. polyphemus horseshoe crabs. Active efforts such as the Just Flip ‘em and Backyard Stewardship programmes by the Ecological Research and Development Group (ERDG) are also contributing to horseshoe crab preservation in the US. There is, however, an absence of protective measures in Asia, where numbers of T. gigas and T. tridentatus have dropped substantially. To better manage these resources and to avoid being reliant on finite supplies of LAL/TAL for endotoxin testing, an alternative assay should be considered.
An alternative test
The recombinant factor C (rFC) PyroGene Assay developed by Lonza presents a sustainable alternative to LAL and TAL assays (see Figure 1). This assay has been recognised by both European Pharmacopoeia (EP) and the Food and Drug Administration (FDA) as an alternative endotoxin test. Factor C, a factor present in horseshoe crab blood, is a key component of LAL and TAL assays and a synthetic version of this factor is utilised in the rFC PyroGene Assay. Using a component functionally and structurally equivalent to that used in LAL/TAL assays, it should be easy for manufacturers to adopt this new test.
If horseshoe crab populations are to be protected in the future, it will be necessary for pharmacopoeia to discuss the definition of this test as an alternative method
Being classified as an alternative test, adoption of the rFC PyroGene Assay requires users to conduct additional validation tests that cost time and money – although by following the protocols supplied by test vendors, it is possible to complete validation procedures in less than three days. Unfortunately, this may still dissuade some from adopting the test, so if horseshoe crab populations are to be protected in the future, it will be necessary for pharmacopoeia to discuss the definition of this test as an alternative method.
The rFC test has additional benefits over LAL/TAL assays, such as ease of use, enhanced endotoxin specificity, statistically robust spike recovery and lot-to-lot consistency. It is hoped that these benefits, as well as the avoidance of affecting horseshoe crab populations, will encourage large pharmaceutical manufacturers to adopt this test. If they do, smaller scale manufacturers are likely to follow suit.
Another challenge facing the endotoxin testing community is low endotoxin recovery (LER), which occurs when endotoxins are added to test samples and are not subsequently recovered, leading to false negative results and questionable test reliability. The problem of inhibition has been seen in similar tests by test vendors in the past and they have usually solved the problems successfully. There is widespread awareness of LER among test vendors, regulators and pharmaceutical manufacturers but the mechanisms behind LER and ways to deal with this problem remain unresolved.
Circumventing low recovery
Fortunately, researchers are working on the task of unravelling the mechanisms behind LER so that sample preparation procedures or the tests themselves might be adapted to overcome it. Research conducted by Johannes Reich at the University of Regensburg suggests that endotoxins are masked by excipients used in some drug formulations.
Endotoxins are lipopolysaccharides (LPS) that naturally aggregate in water due to their hydrophobicity at one end and hydrophilicity at the other. These aggregates are required for LPS detection, but some excipients appear to interfere with endotoxin aggregate formation by reducing the LPSs to monomers that are compartmentalised by micelles when surfactants found in some drug formulations are present. This research suggests that by adapting assay conditions such as pH and magnesium and calcium concentration, and/or by adding polyanionic dispersants such as Lonza Pyrosperse, it might be possible to favour aggregate formation over monomers.
The combination of polysorbate with citrate or phosphate buffers seems to be a particular trigger for LER and manufacturers may consider circumventing the need to unmask LPS by avoiding these combinations. This is, however, an unattractive option for existing products that would require changes to formulations. In this case, introducing additional sample treatment steps might be a better option.
Some manufacturers are avoiding the demasking protocol because it makes the tests they conduct more complex, potentially delaying their product release with substantial financial consequences
Some manufacturers are avoiding the demasking protocol because it makes the tests they conduct more complex, potentially delaying their product release with substantial financial consequences. However, ignoring this issue of LER could lead to problems with approval of new life-saving drugs, including biologics such as monoclonal antibodies. Delays to validation could also have practical consequences for biological products since these products often require a fast turnaround time due to their short shelf lives.
Research such as that discussed should help to overcome LER but this remains a significant problem in endotoxin testing that is recognised by regulatory authorities.
Improvements in efficiency could help the industry meet the growing demands for endotoxin tests. Automation is already in place for some large-scale users but it remains too expensive for many others and unsuitable for testing that is not undertaken on a large scale. As well as improving efficiency, automation has the potential to move endotoxin testing from quality control (QC) laboratories to manufacturing floors, which could allow earlier detection of problems, leading to reduced cost and reduced risk to the public. Limited success of this has been seen so far, but testing on the manufacturing floor could become much more common in the near future.
One risk with automation is that if pharmacopoeia guidelines are slow in progressing with guidelines for the rFC test, automated techniques might be developed for LAL/TAL assays that become obsolete in the near future. By keeping abreast of the latest test technology, pharmacopoeia can assist the producers of automation technology in making equipment that is appropriate or adaptable for new endotoxin testing assays.
Great changes in pharmaceuticals are occurring including, but not exclusively, the growth of biological compounds. The nature of these compounds is such that more detailed and product-specific risk assessment guidelines will need to be produced for them. This is likely to affect endotoxin testing, and the industry and regulators will need to collaborate to promote rapid and mutual progression throughout the endotoxin testing community.
To develop endotoxin tests and guidelines that will meet future demands while minimising the impact on horseshoe crabs, regulatory bodies, pharmaceutical manufacturers, reagent vendors, academic researchers and conservationists will all need to work together
These stakeholders, as well as horseshoe crab conservationists, will all profit from ensuring that innovative methods that improve upon current endotoxin assays are incorporated into the pharmacopoeia and are implemented in the QC laboratories. Change will need to be embraced and to do this a responsive system that ensures improved assays are promptly adopted while maintaining the most important aspect – high standards of patient safety – are in place.
To develop endotoxin tests and guidelines that will meet future demands while minimising the impact on horseshoe crabs, regulatory bodies, pharmaceutical manufacturers, reagent vendors, academic researchers and conservationists will all need to work together. Fortunately, challenges such as LER are being tackled by researchers to protect public health.
Alternative tests have the potential to save horseshoe crab populations but can also provide other advantages over current tests, such as increased reliability. The benefits of these tests can only be obtained with current regulatory guidelines if the pharmaceutical industry is encouraged to conduct the required validation tests necessary for alternative testing. Promoting these alternatives will ensure that new equipment for automation will be compatible with them, so that testers can improve efficiency in their QC process.
All of these developments will be further discussed in the upcoming Global Endotoxin Testing Summit being held by Lonza, 23-25 May 2016. For further information, or to register, see the event website www.lonza.com/endosummit.
Just Flip ‘em and Backyard Stewardship programmes are registered trademarks of the Ecological Research and Development Group Inc. PyroGene and Pyrosperse are registered trademarks of Lonza