A strategic approach to optimisations of testing bacterial endotoxins

Guest, Miriam; Duncanson, Phil; Capper, Karen; Wong, Dennis

article

A strategic approach to optimisations of testing bacterial endotoxins

Given industry’s recent focus on the sustainability of bacterial endotoxin testing (BET), here, AstraZeneca colleagues Miriam Guest, Karen Capper, Dennis Wong and Phil Duncanson share how they worked to establish a short-, mid- and long-term strategy to optimise BET across the global enterprise. They also explore some of the short-term benefits already realised through the company-wide rollout of their work.

The topic of bacterial endotoxin testing (BET), while a unique requirement for the pharmaceutical industry, has far‑reaching impact. In recent years, articles on the reliance on the horseshoe crab within the pharmaceutical industry^1-3 and considerations for alternative testing strategies have been widely discussed.^4-7 Here, the approach taken at AstraZeneca to optimise test methodology to provide assurance of patient safety, reduce the burden on a natural resource and continue to improve in this area is shared.

The endotoxin test typically utilises the lysate refined from the blood of the horseshoe crab; this article addresses the approaches AstraZeneca has taken to minimise the burden on this natural resource. This work considers the United Nations (UN) Sustainable Development Goals (SDG), as well as the importance of change management in a heavily regulated laboratory environment, to provide support and ensure patients receive safe and efficacious products.

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Sustainable development and endotoxin testing

The bacterial endotoxin test has featured in both academic and industry journals over recent years, as industry acknowledged the potential impact of using a natural resource and attempted to mitigate or minimise it. There is a range of approaches that can be taken, from doing nothing, to actively progressing towards a solution.

The UN SDGs⁸ were published in 2015 to provide a clear framework to support the transformation of our planet. They were defined as a call for action to promote prosperity while protecting the planet. The 17 goals highlight both the complexity and the interconnections between education, health, social protection and environmental protection; everybody can contribute in some way to provide a positive means of tackling climate change. Refer to Figure 1 on how some of the specific goals link to endotoxin testing.

Figure 1: UN SDGs linked to bacterial endotoxin testing.

AstraZeneca’s focus on sustainability is fundamental to its company strategy.⁹ Progress is made and monitored across three interconnected sustainability initiatives – increasing access to healthcare, protecting the environment and demonstrating our commitment to ethics and transparency. Efforts are underpinned by our commitment to the UN SDGs and key partnerships at global and local levels.

With the news that the IUCN Red List Index had labelled the Atlantic horseshoe crab as “vulnerable” in 2016, AstraZeneca embarked on a journey to further understand the impact of BET within the global company and built a short-, mid- and long-term strategy to mitigate the impact”

The International Union for the Conservation of Nature (IUCN)¹⁰ brings together experts from across the world with a focus on global conservation. It was established in the 1940s to encourage international co-operation on conservation action. The threats to biodiversity are well documented and the earth is losing species at an alarming rate. Some of the work performed by the IUCN considered the environmental impacts on habitats, as the loss of good quality and good size habitats is a risk to many species. The Union publishes the “IUCN Red List of Threatened Species™” providing data, summarised by experts on the population status of a range of species. This is well established as the most comprehensive inventory of the global conservation status of plant and animal species, and is reviewed and updated as data becomes available.

Considerations for the UN SDGs and the IUCN Red List are key factors when reviewing strategies for BET.

The tests established in the 1970s and 1980s are based on Limulus amoebocyte lysate (LAL), which is produced from the blood of the horseshoe crab. While change in the good manufacturing practices (GMP) of the pharmaceutical microbiology laboratory is difficult, a number of advances have been made in this space in recent years.

BET at AstraZeneca

The first steps in the global project to improve how bacterial endotoxin testing is performed were to understand the existing testing landscape.

Through the global network of microbiology laboratories, a series of engagement sessions were performed. These included virtual meetings and benchmarking surveys, as well as some face-to-face laboratory visits that occurred to further understand the daily challenges that may be addressed in an optimisation programme.

Sites were using a range of different endotoxin techniques in accordance with United States Pharmacopeia (USP) <85>¹¹ or European Pharmacopeia (Ph. Eur.) 2.6.14¹² (and regional pharmacopoeias). Some sites had single testing options while others had multiple options (depending on the portfolio of products at the sites involved). These included:

Gel-clot testing
Turbidimetric and chromogenic microplate
Automated chromogenic microplate
Microfluidic cartridge
Turbidimetric and chromogenic tube readers.

The data indicated that testing a diverse range of products would need more than one testing solution in the toolkit.

During the landscaping activities, the amount of time taken to perform testing was assessed. The review of the workflow considered data entry and approval steps as well as the testing.

A review of invalid test rate assessments produced some interesting results. Sites were requested to monitor and report the number of invalid tests over the course of three months. The data was variable between sites; those sites with the lowest number of invalid test results used automated chromogenic microplates and microfluidic tests. The highest number of invalid tests were observed in the sites doing manual preparation of the microplate. Invalid test rates were reported as less than two percent and up to 20 percent of samples tested – refer to Figure 2 (note data is based on seven sites due to data availability during benchmarking).

Figure 2: Snapshot of invalid tests across seven sites in 2018.

A clear picture of the testing landscape, inefficiencies and challenges was being recognised.

As aspects of sustainability were deemed a key business driver for laying the foundation of the changes, an understanding of lysate wastage was sought. To investigate this, sites were requested to share the number of tests they had reported. This was then compared with the volumes of lysates purchased across the same time period to provide a ‘lysate per test in ml’. (While the volume of lysate per vial may be slightly variable, to ensure the calculation was not overestimated, the lowest volume of lysate was used).

Understanding the sites with the lowest lysate per test figures provides a clear indication of mechanisms to reduce the burden on the horseshoe crab.

Sites with high invalid sample rates also resulted in high ‘lysate per test’ volumes. Refer to Figure 3 for a snapshot (note data is based on seven sites only due to data availability during benchmarking).

Figure 3: Lysate volume (ml) per reported test result across seven sites in 2018 for testing bacterial endotoxins.

The lysate per test values provided a clear insight into the wastage associated with the endotoxin test, signalling an area for improvement should a change be initiated.

For any sustainable microbiology laboratory, the cost of goods is an important consideration. A review was made of the cost of goods, considering all the auxiliary components required for endotoxin testing and a review of the global landscape.

External testing landscape

At the time of project initiation, discussions with providers of endotoxin testing solutions began. This looked across a range of suppliers and their diverse testing options, considering the “available now” technology and the future landscape with the opportunity to consider solutions for a strategy that would encompass short-, mid- and long‑term actions. The roadmap of testing solutions was considered, with the overall aims to meet the business drivers:

Reduce the burden on the horseshoe crab
Reduce the number of invalid samples
Reduce waste
Increase efficiencies
Minimal change burden.

The regulatory landscape was assessed, and international pharmacopoeias had variation in the test methods described for monitoring bacterial endotoxin testing at that time.

With these considerations, the data available within AstraZeneca, data from publications, information shared by technology providers and industry peers; testing options were identified and prioritised for evaluation in the Pharmaceutical Technology & Development Laboratory. The evaluations considered the previously discussed range of business drivers. The laboratory is co-located within a busy QC testing laboratory, which provided a unique opportunity for engagement and feedback from routine users of this technology.

Additional assessments of the available technology were performed within the laboratory to understand the opportunities for change and the reliability of testing options. These technology evaluations considered performance, reliability and the practical aspects of what could potentially be day‑to-day operations.

Roadmap to change

The roadmap was defined thus: the microfluidic testing option would provide an approach to make significant progress in addressing the business drivers in the short term.

The focus would be on transferring all testing of pharmaceutical waters within AstraZeneca to the microfluidic test option. This test had an established history at one site, which provided extensive insight into performance of the test.

Global playbook

To support an efficient network rollout, a global deployment strategy was required. A global implementation guide for sites undergoing installation was established – this provided sites with comprehensive technical information on the proposed change, supporting information for the site-specific business case, results from establishment studies and details of the validation procedures.

It was necessary to align the deployment guide using mandated global standards and quality procedures along with templated examples for validation documentation and risk assessments. As this was considered platform technology, it helped reduce the amount of effort for documentation and ensure sites were optimised with their local implementation processes.

Several other benefits were realised when adopting the global strategy, including the ability to take lessons learnt to avoid multiple sites experiencing common issues and de-risking subsequent deployments, making them quicker and more efficient. During the initial site deployment, several software ‘bugs’ were identified, which were considered unacceptable from a data integrity perspective, as users could be allowed to alter the audit trails. The global project team worked with the vendor to resolve the issue, removing this risk for subsequent sites adopting the new technology.

Site rollout continued over the course of two years, with some challenges being observed on the journey, including the pandemic restricting travel. Novel ways of working were sought, including utilising technology for remote installation and vendor qualification taking place remotely using virtual Windows viewer sessions and HoloLens technology.

Short-term objectives met

The short-term objectives to significantly reduce the burden on the horseshoe crab have been achieved. Testing of waters has transitioned and efficiency benefits have been appreciated by the sites. The reduction in waste has also led to a reduction in cost of goods.

The overall lysate consumption has been reviewed and comparing 2016 data with 2021 data on lysate purchased, reveals the total volume has reduced by more than 60 percent.

Moreover, a review of the hands-on testing time demonstrated a reduction of over 3,000 hours across sites.

An additional efficiency benefit was realised, as training time for new users was reduced.

Mid-term strategic planning

To further reduce the burden on the horseshoe crab, the mid-term plan considered two aspects: the potential to test samples other than pharmaceutical waters on the microfluidic platform and also to generate data in the Pharmaceutical Technology & Development Laboratory on synthetic alternatives.

Experimental work has been executed on a challenging test matrix; a group of products that typically cause inhibition in routine testing and was selected as a high dilution factor is required. Use of the microfluidic cartridge has demonstrated that the dilution factor can be reduced from 1:1000 to 1:400 when comparing traditional LAL techniques with the cartridge, which provides more valuable indication of a contamination event. In addition to the inhibition/enhancement screening, samples were spiked with parallel LAL water controls at 200 Endotoxin Units (EU)/ml Reference Standard Endotoxin to evaluate possible storage-related masking effects. The spiked test sample and control were held in ambient conditions and tested at Time 0, Day 1 and Day 7. The recovery of endotoxin from the test article was compared to the control sample for each timepoint (refer to Table 1).

Table 1: Spiked endotoxin recovery over time

To evaluate the robustness of synthetic alternatives, several samples with stable background endotoxin have been obtained; this includes rejected articles where aqueous hold times were extended beyond use, resulting in elevated endotoxin reported and also samples of technical material with low levels of background endotoxin. Performing a robust validation exercise provides an opportunity to further de-risk future use of the test.

The external landscape is continuously reviewed, including attendance at external forums, events and meetings on the topic, as well as literature reviews. The strategy is fluid and can be adapted when new data or new technology becomes available.

Long-term strategic planning

On completion of a successful validation of a synthetic alternative, product testing for the development portfolio will be considered. This may result in this testing strategy being included in clinical trial applications, but ultimately the aim is to have that data available for review in a new drug application.

Further considerations

Consolidating the information available on horseshoe crab populations is an activity that does not stop; there is always progress and new information available. AstraZeneca is committed to reviewing the data and responding to the challenging landscape. The picture is complex and single activities do have an impact; however, a holistic strategy is critical for the survival of this species.

Benefits of strategic approach to optimise testing bacterial endotoxins

A range of options are available to optimise the endotoxin test; fundamentally, multiple test solutions are required to meet the demands of a complex portfolio of injectable products.

Considerations to reduce the burden on the horseshoe crab have been factored in, alongside other impacts of the test and the day-to-day activities of the busy QC microbiology laboratory. Global acceptance and the need to provide assurance of patient safety were other key areas driving decisions, alongside published data on available tests, in-house generated data, historical knowledge of the test platforms and an eye on the future portfolio.

Using microfluidics has enabled a range of benefits to be realised by AstraZeneca in a short space of time.

Using microfluidics has enabled a range of benefits to be realised by AstraZeneca in a short space of time. A review of the overall picture of endotoxin testing across the network demonstrates a greater than 60 percent reduction in the volume of lysate purchased and a saving of more than 3,000 hours.

The challenges on the horseshoe crab populations are complex and not limited to the biomedical industry. Healthy habitats for populations are vital for the success of the species.

There are several approaches that support reducing the burden on the horseshoe crab and the pharmaceutical industry has a responsibility to help deliver the UN SDGs.

Building a strategic approach with a holistic view of short- and long-term improvement projects can lead to significant benefits.

About the authors

Miriam Guest is a Principal Microbiologist at AstraZeneca, working in the New Modalities and Parenteral Development group; based at their Macclesfield site in the UK. Miriam has worked in pharmaceutical development for over 20 years, in both microbiology and formulation laboratories and GMP manufacturing environments; more recently supporting complex supply chains and designing robust control strategies, including the associated regulatory authoring and interactions. She leads the AZ Global Microbiology Forum, bringing together the global network of microbiology labs to drive standardisation and quality principals in microbiology. Miriam devised and leads AstraZeneca’s “21st Century Microbiology Strategy”, to innovate, industrialise and implement technology solutions to drive efficiencies and process robustness benefits within the AZ global network. She is an active committee member of the Pharmaceutical Microbiology Interest Group (Pharmig) and is involved in various cross industry collaborative groups.

Dennis Wong has worked in BioPharma Quality for over 20 years in a range of roles, including lab management (microbiology and biosafety testing), analytical technology transfer and new technology deployment.
In his current role Dennis is responsible for the deployment of new technology and digital platforms across the QC network and supports the standardisation of business processes for global QC.

Karen Capper is a pharmaceutical microbiologist with over 30 years’ experience in microbiology positions within the pharmaceutical industry. She has extensive knowledge in managing and operating QC microbiological and analytical laboratories as well as manufacturing experience in aseptic production. She has specialist knowledge in the microbiological assurance of aseptic processes, environmental control of non-sterile pharmaceuticals and API facilities. She currently leads the Global Microbiology Team in Product Development and is responsible for the microbiological assurance and testing of the products in development by AstraZeneca. Karen is also a course tutor for the PMAT Course at Manchester University where she tutors the GMP in Microbiology Laboratories and Antimicrobials units.

Phil Duncanson, PhD is a microbiologist with over 20 years’ experience in the clinical, industrial and pharmaceutical industries. He has extensive knowledge in managing and operating QC microbiological and analytical laboratories. The majority of Phil’s pharmaceutical experience has been gained at sterile drug product manufacturing sites for both synthetic and biologic products, where he gained experience in aseptic manufacturing from manufacturing and quality assurance perspectives. He is currently the Business Process Owner for Microbiology in Quality and is responsible to strategy, standardisation and issue management at AstraZeneca.

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