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Cathy Olsen, PhD | Sr. Applications Scientist | Molecular Devices
Timothy Bolus | Sr. Compliance Program Manager | Molecular Devices
Delaney Novak | Field Applications Scientist | FUJIFILM Irvine Scientific, Inc.
Timothy Francis | Sr. Field Applications Scientist | FUJIFILM Irvine Scientific, Inc.

Introduction

Monitoring for contaminants is a critical step during the production process in the pharmaceutical and medical device industries. Endotoxin, a frequent contaminant that is found in the cell wall of gram-negative bacteria, can cause fever, inflammation, headache, nausea, and even death. Therefore, careful testing of all medical devices and injectable pharmaceuticals is required to ensure there is an endotoxin concentration less than the endotoxin limit of the product. The sensitive and specific Limulus amebocyte lysate (LAL) assay is very often the test of choice for detecting bacterial endotoxin. When a sample containing endotoxin is added to LAL (obtained from the horseshoe crab Limulus polyphemus), an enzyme-mediated cascade is triggered, resulting in an increase in clotting that can be detected qualitatively as a gel clot in a test tube, or quantitatively via turbidimetric or colorimetric microplate- based methods.

Here, we demonstrate the performance of two bacterial endotoxin tests from FUJIFILM Irvine Scientific, Inc. PYROSTAR™ ES-F/Plate and Limulus Color KY Test, in combination with Molecular Devices SpectraMax® Microplate Readers. With the PYROSTAR ES-F/Plate, LAL coagulation is monitored as an increase in turbidity over time, measured kinetically with an absorbance microplate reader. The more endotoxin is present in a test sample, the shorter the onset time, or time required for the absorbance of a sample to increase above its initial value by a specified amount. A standard curve of onset time versus concentration of a control standard endotoxin (CSE) is performed so that the amount of endotoxin present in a test sample can be interpolated from it. The PYROSTARES-F/Plate assay can be used to detect levels of endotoxin from 0.01 to 10 EU/mL. The Limulus Color KY Test utilizes a similar workflow but is a colorimetric method enabling even more sensitive endotoxin quantitation, from 0.0005 to 5.0 EU/mL.

With Molecular Devices SoftMax Pro software, the PYROSTAR ES-F/Plate and Limulus Color KY assays can be run using preconfigured protocols, available through the Spectranet Customer Care Portal, with all the required instrument settings as well as complementary data analysis and automatic reporting of results. SoftMax Pro GxP Software is the latest, most secure software to achieve full FDA 21 CFR Part 11 and EudraLex Annex 11 compliance with streamlined workflows to ensure data integrity.

Materials

• PYROSTAR ES-F/Plate reagent
  (FUJIFILM Irvine Scientific, Inc. cat. #543-10331)
• Limulus Color KY reagent
  (FUJIFILM Irvine Scientific, Inc. cat. #291-53101)
• Control Standard Endotoxin (CSE, included with PYROSTAR ES-F/Plate and Limulus Color KY Reagents)
• LAL Reagent Water
  (LRW, FUJIFILM Irvine Scientific, Inc. cat. #LRW-00125)
• Endotoxin-free microplates
  (BioClean Plate Wako, cat. #293-35221)
• Endotoxin-free pipettes
• Endotoxin-free dilution tubes
• SpectraMax ABS Plus Microplate Reader (Molecular Devices)
• Additional SpectraMax readers with absorbance detection mode and kinetic reading (provide equivalent results to that shown for SpectraMax ABS Plus):
  • SpectraMax M5e Multi-Mode Microplate Reader
  • SpectraMax Mini Multi-Mode Microplate Reader
  • SpectraMax i3x Multi-Mode Microplate Reader
  • SpectraMax iD5 Multi-Mode Microplate Reader

Methods

The SpectraMax ABS Plus reader was turned on, and the reader temperature was set to 37°C through SoftMax Pro software.

PYROSTAR ES-F/Plate reagent, or Limulus Color KY Test solution, was reconstituted by adding the volume of LAL Reagent Water (LRW) indicated in the product insert. The vial was swirled gently to dissolve the lyophilized reagent, avoiding bubbles. Control Standard Endotoxin (CSE) was reconstituted to 1000 EU/mL by adding the volume of LRW required, based on information provided on the Certificate of Analysis for the LAL and CSE matched kit. The vial was vortexed for two minutes to reconstitute thoroughly. Unused reconstituted CSE was stored at 4°C for up to one month and was vortexed for one minute just before each further use.

*Slow carriage speed for the Limulus Color KY assay is optional.

Table 1. Instrument settings used to acquire data for the PYROSTAR ES-F/Plate and Limulus Color KY test endotoxin assays.

A 1:10 dilution series of CSE for the standard curve was prepared in LRW. For the PYROSTAR ES-F/Plate kit, standards ranged from 0.01 to 10 EU/mL; for the Limulus Color KY test, 0.0005 to 5 EU/mL standards were used.

A sample of tap water was collected to represent a sample containing naturally occurring endotoxin. Products can be appropriately diluted as needed to bring the endotoxin limit of the product within the range of the standard curve and mitigate the risk of product interference. In this case, the sample was diluted 2-fold.

A positive product control was prepared by spiking the test sample to the midpoint concentration of the standard curve for each assay. The purpose of this control is to confirm that the sample does not introduce any interfering factors and that the result is valid. Inhibition and enhancement types of interference can cause false negatives or false positives in an assay. Certain samples, such as proteinaceous ones, are more prone to interference than others, and interference must be mitigated with dilution or a sample-specific treatment before using a LAL assay.

To set up the assay plate, 50 μL of LRW was added to wells designated as negative controls, and 50 μL of CSE standard dilutions were added to triplicate wells. 50 μL of sample dilution and corresponding positive product control were added to designated wells. After each of the solutions were added to the plate, either 50 μL PYROSTAR ES-F/Plate reagent or 50 μL of Limulus Color KY test solution was added to all assay wells using a multi- channel pipette. The plate was placed in the pre-heated reader, and the reading of the reaction was initiated. For each test, a preconfigured, assay-specific protocol in SoftMax Pro software was used. To accurately run each assay, these protocols are configured with the parameters outlined in the ES-F/Plate and Color KY package inserts, which are shown in Table 1, as well as complete data analysis features so that results are viewable immediately upon completion of the plate read. Progress could be monitored by viewing kinetic traces in the plate section of the protocol.

For all SpectraMax readers listed in Materials, the Limulus Color KY test produced data similar to those obtained with the SpectraMax ABS Plus reader. Results with the PYROSTAR ES-F/Plate assay benefited from use of the Slow Carriage Speed feature, selectable in SoftMax Pro software for the SpectraMax ABS Plus and SpectraMax M5e readers.

Results

PYROSTAR ES-F/Plate

Kinetic traces showing onset times for standards in the PYROSTAR ES-F/Plate assay are shown in Figure 1. Mean onset time for each standard was plotted vs. CSE concentration by SoftMax Pro software (Figure 2).

Figure 1. Kinetic traces of representative standards for the PYROSTAR ES-F/Plate assay. Vertical red lines indicate onset times (onset OD = 0.015).

Using a protocol in SoftMax Pro software that facilitated analysis of data and presentation of results, multiple assay parameters including product dilution test, positive product control spike recovery %, product (sample) pass/ fail, and overall test result, were automatically displayed based on user-input values and calculations performed by the software. These results were summarized in the Product Summary section of the protocol, where the overall validity or invalidity of the test (depending on whether the sample passes specifications, along with other calculated parameters) is easily reviewed (Figure 3).

For the example shown, the r value for the standard curve (> 0.98), product dilution test (dilution factor was less than the maximum valid dilution), and PPC recovery (between 50 and 200%) all passed requirements for the test used. However, as the endotoxin concentration in the sample at 2.005 EU/mL exceeded the sample endotoxin limit of

0.500 EU/mL, the overall judgment in the summary section alerted the user to the invalidity of the result.

Figure 2. Endotoxin standard curve for the PYROSTAR ES-F/Plate assay. Mean onset time (seconds) vs. endotoxin standard concentration was plotted using the log-log curve fit in SoftMax Pro software (n = 3, R2 = 0.979).

Figure 3. Product Summary section of the PYROSTAR ES-F/Plate protocol in SoftMax Pro software. Results are indicated as pass or fail, and the Overall Judgment of the test here is indicated as Invalid due to the failure of the product to meet test specifications.

Limulus Color KY test

Kinetic traces showing onset times for standards in the Limulus Color KY assay are shown in Figure 4. Mean onset time for each standard was plotted vs. CSE concentration by SoftMax Pro software (Figure 5).

As with the previously described assay, a specially configured SoftMax Pro software protocol was used to ease analysis and clearly present the results. The r value, product dilution, and positive control all passed requirements for the endotoxin test. The product (sample) endotoxin concentration exceeded the specified endotoxin limit, and the overall judgment was therefore indicated as “FAIL” in the protocol’s product summary section (Figure 6).

Figure 4. Kinetic traces of representative standards for the Limulus Color KY assay. Vertical red lines indicate onset times (onset OD = 0.015).

Figure 5. Endotoxin standard curve for the Limulus Color KY assay. Mean onset time (seconds) vs. endotoxin standard concentration was plotted using the log-log curve fit in SoftMax Pro software (n = 3, R2 = 0.998).

Figure 6. Product Summary section of the Limulus Color KY protocol in SoftMax Pro software. Results are indicated as pass or fail, and the Overall Judgment of the test here is indicated as Invalid due to the failure of the product to meet test specifications.

Conclusion

The Limulus Amebocyte Lysate (LAL) test, recognized by the U.S. Pharmacopeia (USP), European Pharmacopeia (Ph. Eur.), and Japanese Pharmacopeia (JP), is a mainstay of drug and device testing for bacterial endotoxins. The quantitative, kinetic PYROSTAR ES-F/Plate and Limulus Color KY assays offer labs reliable methods for ensuring safe endotoxin levels, detecting concentrations as low as 0.01 EU/mL or 0.0005 EU/mL, respectively. The SpectraMax ABS Plus, as well as SpectraMax multi-mode readers, with SoftMax Pro software, provide a streamlined workflow with automatic calculation of results using preconfigured protocols. There is also a specially configured protocol for each assay available for use in regulated labs. The SoftMax Pro GxP Software provides a system audit trail with e-signature statement capabilities and an audited document workflow within a SQL database.

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