By Tim Sandle, Ph.D.
The USP and European Pharmacopoeia (EP) require testing for bacterial endotoxin in pharmaceutical products, water, intermediates, and medical devices based on the Limulus amebocyte lysate (LAL) assay (or Tachypleus under the Japanese pharmacopeia).1 In Europe, the options have been expanded to include recombinant lysate through method verification, although method validation is required for U.S. adoption. The screening of many types of pharmaceutical products for endotoxin remains an important release test because contaminated products could cause fever, inflammation, and endotoxemia in patients.2
Despite having been a pharmacopeia method since 1985 (and possibly, depending upon the definition applied, the first rapid microbiological method), the assay is subject to variability, and it contains several manual steps that lend themselves to automation.
Ideas for extending automation to the endotoxin test have been active since the 1980s (when platforms were designed to automate parts of the gel-clot assay),3 including the use of robotics to remove some of the repetitive tasks carried out by analysts. This has primarily been approached through machinery, to improve assay precision, and through other forms of automation to improve data integrity in terms of sample traceability (including data transfer to laboratory information management systems) and to enable the assessment of data patterns and trends through software. Another key innovation is using online endotoxin testing to enable real-time assessment of pharmaceutical water systems, although this cannot replace point-of-use assessments of water quality. In recent years, some commercial solutions have become available. This article outlines the key criteria when seeking a robotic endotoxin detection system.
Bacterial Endotoxin Test Variation
With biological tests, all measurements susceptible to variations in analytical conditions should be suitably controlled as far as is practicable. A distinction should be drawn between technical variables and biological ones, with the former being easier to control. Here, the LAL assay has a relatively high level of variability, even for a biological assay.4 This variation is due to three principal factors: reagents, the product tested, and issues inherent in the method. Examples of sources of variation include:5
- pipetting technique and pipettors (pipettes must be regularly assessed for accurate and repeatable functioning)
- aseptic technique
- vortex mixing
- Accuracy of timing
- temperature control
- control of external vibrations and other aspects of the laboratory environment
- analyst capability
- calibration of instruments
- control of storage conditions
- quality of assay reagents.
Many of the above are either directly reliant upon human involvement or can be influenced by lapses; hence, a human factors approach to designing more reliable solutions can decrease both the assay variability and mistakes (Type 1b and Type 1a factors in out of specification terminology).
Reproducibility and precision are among the essential assay criteria for endotoxin testing. The precision of any analytical procedure is important and generally defined as the degree of agreement among individual test results (or, in assay terminology, the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single, homogenous volume of the biological matrix).6 It is therefore a useful and important part of quality control. The coefficient of variation is often a useful measure of assay precision.
Selection Of Robotic Systems
The following framework can aid in the decision-making process. General requirements of an automated robotic platform are:
- Does the robotic testing tool meet the project requirements?
- Has the software been developed for the specific technology?
- What is the level of usability of the automation testing tool?
- What is the footprint of the automated platform relative to laboratory space?
- Are any specific environmental controls required?
- How are samples identified and traced (bar coding)?
- What is the relative cost per test?
- How reliable is the automated solution?
- What is the sample throughput?
- What will be required to validate the instrument and to gain regulatory approval?
- What are the maintenance requirements?
- What are the options when the platform breaks down or is offline?
- What level of analyst training will be required?
- What experience does the vendor of the automation testing tool have in providing technical support?
- How adaptable and scalable is the platform to meet future expansion?
- What level of engineering support is required?
In addition, for an endotoxin solution, you will need to consider the aspects that you want automated and that can be automated. This may include:
- consumable traceability
- consumable preparation
- LAL reagent traceability
- LAL reagent preparation
- sample traceability
- sample preparation
- software preparation and configuration
- temperature control and incubation
- sample reading (and reading intervals)
- end of test reading and assessment
- sample disposal.
For those using plate or cartridge systems, the transfer over to a robotic system will be less of a conceptual transfer than for those users switching from tube readers, given that the tube reader does not readily adapt to the automation process. You also need to consider the number of workflows that will be required and how straightforward they are to configure in relation to different product types.
Consider specific aspects of an automated endotoxin testing platform:
- Is an endpoint or kinetic system required?
- Are all of the sample types currently assayed suitable for the new platform?
- This can be a specific concern with samples that are turbid or of different colors.
- A related consideration includes applications where high potencies of endotoxin are required, such as when performing depyrogenation studies.
- Can the automated pipetting functionality cope with samples of different viscosities?
- What is the degree of analyst involvement in the conventional workflow compared to the robotic system workflow?
- Can, for example, different sample matrices be run for when a sample needs to be assayed at more than one dilution, especially in cases where test inhibition or enhancement is likely?
- How many samples per test can be accommodated?
- How is the system protected against contamination?
- Consider, for example, the barrier around the platform, whether the platform is protected by a localized airflow, etc.
- What is the preparation time and run time?
- Some systems may require additional hold periods for samples or reagents, and this may require qualifying.
- Are standard curves preloaded into the software, preprepared on a test card, or produced as part of sample preparation?
- This consideration leads to whether control standard endotoxin is directly handled by the system or whether it is preloaded into a test plate.
- If a naturally occurring endotoxin standard is required, can this be accommodated?
- How are product positive controls prepared?
- How are negative controls prepared?
- What is the sample throughput?
- How much sample is required?
- Some systems reduce the sample volume required. Here, care must be taken in terms of assessing the limit of quantification, which is the lowest analyte concentration that can be quantitatively detected with a stated accuracy and precision, as this may not be equivalent when comparing conventional and robotic systems.
- A further consideration is that some assays require a minimum quantity of the active ingredient for a reliable estimation, especially with proteinaceous products. Therefore, dilute protein solutions may require a larger volume to reach the limit of detection, albeit a volume that does not interfere with the endotoxin assay.
- On the other hand, using reagents in smaller, concentrated volumes can limit freeze/thaw cycles and minimize the risk of introducing contamination into reagent stocks.
- What is the time-to-result?
- How does the time-to-result relate to the required test sensitivity?
- How much reagent is used and what is its cost?
- How much consumable is used and what is its cost?
- How accurate are the temperature control and test time?
- What is the test sensitivity in terms of endotoxin detection?
- Are all pipetting steps automated?
- Is there a reduction in pipetting steps?
- How are dilutions programed into the system?
- What are the intended and actual accuracy of pipetting? How do these compare with a human analyst?
As well as seeking improved reliability, in terms of test results that are suitably precise and ideally more reproducible compared with the more conventional manual methods, the robotic solution should have equivalent sensitivity (conventionally with endotoxin testing, this is the range 5.0 to 0.005 EU/mL); be able to show any variation between replicates (and achieve a coefficient of variation of below 10%); and produce sufficient linearity when control endotoxin solutions (purified lipopolysaccharide) are assayed. Accuracy will depend on the number of replicates run. The larger the sample size for a particular evaluation, the greater the certainty surrounding the estimate of accuracy.7
It is also useful to understand how the system detects error, including the effect of bubble formation (as might be caused by an external vibrational wave) or in the event of power disruption or when the test is manually stopped.
Another aspect to consider is whether the endotoxin solution is based on the conventional kinetic methods (turbidimetric or chromogenic) or an alternative, such as a fluorescence-based assay. A related aspect is whether both an animal-derived and recombinant lysate can be used.
As with any automated system, a thorough understanding of both the workflow and the data flow is required. The general requirements for data integrity must be met. The recommended period for requalification of both the instrument and the samples tested also needs to be understood. This will include the expected frequency for running control samples.
Regulatory Filing Strategy
At an appropriate stage it must be determined whether a robotic platform meets the requirements of the pharmacopoeia (USP <85>, EP 2.6.14, and JP 4.01) or to what degree the requirements are met in relation to the platform falling under the guidance chapters for rapid and alternative microbiological assays. Such an assessment is necessary for developing the regulatory filing strategy and seeking the appropriate license changes.
This article looks at some of the important criteria to consider when adopting robotic endotoxin solutions. It is important that any alterations to a method are subject to change control and that verification is performed as a side-by-side comparison with the established method and reagents to assess the effect of changes on assay accuracy and precision.
- Williams, K.L. Endotoxins, Pyrogens, LAL Testing and Depyrogenation, 2nd edition, 2007, CRC Press: Boca Raton
- Opal, S. M. et al., (Eds): Endotoxemia and Endotoxin Shock: Disease, Diagnosis and Therapy. Contrib Nephrol. Basel, Karger, vol 167, pp 14-24, 2010
- Tsuji K, Martin PA. Evaluation of robot automated chromogenic substrate LAL endotoxin assay method for pharmaceutical products testing. Progress in Clinical and Biological Research. 1985 ;189:151-167
- McCullough, K.C. and Weider-Loeven, C. Variability in the LAL Test: Comparison of Three Kinetic Methods for the Testing of Pharmaceutical Products, Journal of Parenteral Science and Technology, 1992, 44: 69-72
- Sandle, T. Variability and the LAL Assay for Bacterial Endotoxin Detection, Journal of GxP Compliance, 2019, 23 (5): 1-10
- Kirkland P.D. and Newberry K.M. Your assay has changed – is it still ‘fit for purpose’? What evaluation is required? In Diagnostic Test Validation Science: A key element for effective detection and control of infectious animal diseases (I.A. Gardner & A. Colling, eds), 2021 Rev. Sci. Tech. Off. Int. Epiz., 40 (1), XXYY. doi:10.20506/rst.40.1.XXXX
- Serdar CC et al. Sample size, power and effect size revisited: simplified and practical approaches in pre-clinical, clinical and laboratory studies. Biochem Med (Zagreb). 2021 ;31(1):010502
About The Author:
Tim Sandle, Ph.D., is a pharmaceutical professional with wide experience in microbiology and quality assurance. He is the author of more than 30 books relating to pharmaceuticals, healthcare, and life sciences, as well as over 170 peer-reviewed papers and some 500 technical articles. Sandle has presented at over 200 events and he currently works at Bio Products Laboratory Ltd. (BPL), and he is a visiting professor at the University of Manchester and University College London, as well as a consultant to the pharmaceutical industry. Visit his microbiology website at https://www.pharmamicroresources.com.