Monitoring for contaminants is an essential production step in the pharmaceutical and medical device industries.
Endotoxin is frequently found in the cell wall of gram-negative bacteria, and this contaminant can cause inflammation, headaches, fever, nausea, and even death. Careful testing of all medical devices and injectable pharmaceuticals is, therefore, vital in ensuring that the endotoxin concentration is less than the product’s endotoxin limit.
The limulus amebocyte lysate (LAL) assay is both sensitive and specific, regularly making it the test of choice for bacterial endotoxin detection.
Molecular Devices provides a validated, ready-to-use workflow for kinetic endotoxin testing to support these requirements. This workflow integrates automated data analysis, optimized reader settings, and software designed for use in regulated environments.
This article showcases the performance of FUJIFILM Biosciences PYROSTAR™ ES-F/Plate Test when employed in conjunction with Molecular Devices’ SpectraMax® i3x Multi-Mode Microplate Reader.
LAL coagulation is monitored as an increase in turbidity over time with this assay, measured kinetically with an absorbance microplate reader.
Higher levels of endotoxin present in a test sample result in a shorter onset time, or time necessary for the absorbance of a sample to increase above its initial value by a specified amount, 0.015 OD for this specific assay.
A standard curve of onset time versus concentration of a control standard endotoxin (CSE) is completed to allow the amount of endotoxin present in a test sample to be interpolated. The PYROSTAR ES-F/Plate assay can be used to quantitatively detect endotoxin levels ranging from 0.01 to 10 endotoxin units (EU/mL).
The SoftMax® Pro software from Molecular Devices allows the PYROSTAR ES-F/Plate or other kinetic turbidimetric endotoxin assay to be run using a preconfigured protocol.
This protocol includes all required instrument settings, complementary data analysis, and the automatic reporting of results. This functionality is available in the software’s Protocol Library (version 7.4 or higher) or through the Spectranet Customer Care Portal.
The SoftMax Pro GxP software is the latest, most secure software for use in regulated environments, achieving full FDA 21 CFR Part 11 and EudraLex Annex 11 compliance along with streamlined workflows designed to ensure data integrity.
Materials
- PYROSTAR ES-F/Plate Reagent (FUJIFILM Biosciences, Catalog #543-10331)
- Control Standard Endotoxin (CSE, included with PYROSTAR ES-F/Plate assay)
- LAL Reagent Water (LRW, FUJIFILM Biosciences, Catalog #LRW-00125)
- Endotoxin-free microplates (BioClean Plate Wako, Catalog #293-35221)
- Endotoxin-free dilution tubes
- Endotoxin-free pipettes
- SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices)
- Other SpectraMax readers with absorbance detection mode and kinetic reading can be used with the PYROSTAR ES-F/Plate assay, including:
- SpectraMax ABS Plus Microplate Reader
- SpectraMax M-Series Multi-Mode Microplate Readers
- FlexStation® 3 Multi-Mode Microplate Reader
Methods
The SpectraMax i3x reader was turned on, either via the reader’s touchscreen or via the SoftMax Pro software, to set the reader temperature to 37 °C.
PYROSTAR ES-F/Plate Reagent was reconstituted by adding the volume of LAL Reagent Water (LRW) listed in the product insert. The vial was gently swirled to dissolve the lyophilized reagent and prevent bubbles.
Control standard endotoxin (CSE) was reconstituted to 1000 EU/mL. This was achieved by adding the required LRW volume, based on the information provided in the Certificate of Analysis for the LAL and CSE-matched kit.
The vial was vortexed for two minutes to reconstitute its contents thoroughly. Unused reconstituted CSE was stored at 4 °C for up to one month before being vortexed for one minute just before each further use.
Next, a 1:10 dilution series of CSE was prepared in LRW for the standard curve. Standards ranged from 0.01 to 10 EU/mL.
Table 1. Instrument settings used to acquire data for the PYROSTAR ES-F/Plate endotoxin assay. The 96 Well Standard Slow Motion_i3x* plate type must be imported into the Plate Type Settings in SoftMax Pro Software. It is used to slow the speed of microplate movement in the SpectraMax i3x reader during the kinetic reading; this ensures the clot is not disrupted and prevents aberrant absorbance measurements. Source: Molecular Devices UK Ltd
| Parameter |
Setting |
| Read mode |
Absorbance (ABS) |
| Read type |
Kinetic |
| Wavelength(s) |
Lm1: 405 nm |
| Plate type |
For i3x: 96 Well Standard Slow Motion_i3x*
Other readers: 96 Well Standard |
| Timing |
Total run time: two minutes
Interval: forty seconds |
| Shake |
Before first read: 10 seconds |
| More settings |
Carriage speed: Slow (SpectraMax ABS Plus, M-Series, or FlexStation 3 readers) |
*Available for download on Spectranet, Molecular Devices’ customer care portal
The assay plate was set up by adding 50 μL of LRW to wells designated as negative controls. Next, 50 μL of CSE standard dilutions were added to wells in triplicate. A total of 50 μL of sample dilution and the corresponding positive product controls were also added to the designated wells.
Similarly, 50 μL PYROSTAR ES-F/Plate Reagent was added to all assay wells using a multichannel pipette after each of the solutions had been added to the plate.
The plate was placed in the pre-heated reader before initiating the reaction reading.
The ready-to-use, assay-specific protocol in the SoftMax Pro software was used for data generation and analysis. The protocol is configured with the parameters outlined in the PYROSTAR ES-F/Plate package insert (Table 1) to accurately run each assay.
This instrument also delivers complete data analysis, allowing results to be viewable immediately upon completion of the plate read. Progress was monitored by viewing kinetic traces in the protocol’s plate section.
The PYROSTAR ES-F/Plate assay produced data similar to that obtained with the SpectraMax i3x reader for all SpectraMax readers listed in the Materials section.
Slowing down plate movement or implementing specialized plate movement optimization minimizes clot disruption and enhances kinetic assay consistency for the SpectraMax i3x reader. This plate definition is available via Molecular Devices’ Spectranet customer care portal.
Slow carriage speed is offered as an option in SoftMax Pro software settings for other SpectraMax readers.
Results
Figure 1 features kinetic traces displaying onset times for standards in the PYROSTAR ES-F/Plate assay.
The SoftMax Pro software’s 96 Well Standard Slow Motion_i3x plate type is required for the kinetic turbidimetric endotoxin assay on the SpectraMax i3x reader.
This plate definition effectively reduces the speed of the instrument’s plate carriage during the kinetic plate read, ensuring smoother kinetic data by preventing clot disruption.
Onset times were calculated with the mean onset time for each standard plotted versus CSE concentration using the SoftMax Pro software (Figure 2). The standard curve R2 for the two test runs shown was >0.98, meeting the assay's specified criteria.
Figure 1. Kinetic traces of representative standards for the PYROSTAR ES-F/Plate assay. Vertical red lines indicate onset times (onset OD = 0.015). Data were generated using a ‘slow’ plate definition that ensures smooth kinetic data. Image Credit: Molecular Devices UK Ltd
This approach has been designed to support regulatory compliance via:
- Data integrity: The approach ensures that results are consistently complete, accurate, and protected throughout the assay workflow. This is key to supporting regulatory readiness and confident decision-making.
- Audit trails: They capture a traceable and complete history of data and user actions, streamlining inspections and making quality assurance processes more robust.
- Electronic signatures: Facilitate secure sign-off and user authentication, supporting laboratories’ efforts to meet 21 CFR Part 11 and other regulatory requirements.
- Regulated workflow support: Standardized processes are in place from data acquisition to reporting, minimizing variability while better ensuring compliance with global regulatory standards.
Figure 2. Endotoxin standard curves for two separate test runs of the PYROSTAR ES-F/Plate assay. Mean onset time in seconds, calculated from the kinetic data, vs. endotoxin standard concentration was plotted using the log-log curve fit in SoftMax Pro software. Test 1 (red) R2 = 1.000; test 2 (blue) R2 = 0.990. Image Credit: Molecular Devices UK Ltd
Conclusion
The limulus amebocyte lysate (LAL) test is recognized by the European Pharmacopeia (Ph. Eur.), the U.S. Pharmacopeia (USP), and the Japanese Pharmacopeia (JP), and is a cornerstone of drug and device testing for bacterial endotoxins.
The quantitative, kinetic PYROSTAR ES-F/Plate assay offers labs a dependable means of ensuring safe endotoxin levels, detecting concentrations as low as 0.01 EU/mL.
The SpectraMax i3x reader and the SpectraMax readers discussed can be employed alongside the SoftMax Pro software to deliver a streamlined workflow. Automatic result calculation is implemented via a specifically configured protocol, ideally suited for use in both regulated and non-regulated labs.
The SoftMax Pro GxP software delivers a system audit trail with e-signature statement functionality, coupled with an audited document workflow housed within a SQL database.
The powerful combination of the SpectraMax microplate readers, PYROSTAR ES-F/Plate assay, and SoftMax Pro software offers a scalable kinetic endotoxin testing solution, empowering laboratories to enhance workflow efficiency, generate reliable quantitative results, and support regulatory compliance.
Acknowledgments
Produced from materials originally authored by Cathy Olsen, PhD, and Timothy Bolus from Molecular Devices, and Delaney Novak and Timothy Francis from FUJIFILM Biosciences.
About Molecular Devices UK Ltd
Molecular Devices is one of the world’s leading providers of high-performance bioanalytical measurement systems, software and consumables for life science research, pharmaceutical and biotherapeutic development. Included within a broad product portfolio are platforms for high-throughput screening, genomic and cellular analysis, colony selection and microplate detection. These leading-edge products enable scientists to improve productivity and effectiveness, ultimately accelerating research and the discovery of new therapeutics. Molecular Devices is committed to the continual development of innovative solutions for life science applications. The company is headquartered in Silicon Valley, California, with offices around the globe. For more information, please visit www.moleculardevices.com.
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