APR May/June 2023 - 59

VENDOR VIEWPOINT
The ratio of sample to lysate (1:1) is very important. Most LAL
manufacturers recommend using 100 µL of sample to 100 µL of LAL
for 96-well microplate readers and tube readers. There are some
lysates that utilize less than 100 µL of sample and lysate, and these
are typically run in microplates that have smaller wells. However,
the 1:1 ratio is still maintained. This 1:1 ratio has been proven over
years to accurately deliver results for BET. Using 100 µL of sample and
lysate is not a regulatory or pharmacopeia requirement. Rather, it is a
recommendation on how much volume to use to meet the 1:1 ratio
given the well size in a microplate or tube reader for the desired path
length and absorbance to reach optical density.
Microfluidics enables accurate, reliable, and
compliant endotoxin testing using smaller
reaction volumes
New innovations for LAL testing have emerged in recent years such
as the Sievers Eclipse BET Platform. The Eclipse utilizes microfluidic
technology and centripetal force to accurately deliver small volumes
of liquid to the specific segments on the microplate, thus drastically
reducing the amount of reagent needed and simplifying the setup.
Microfluidics has been a thriving research field for over 30 years due
to its abilities to meet demands for chemical and biological analyses
that are accurate, cost-effective, reliable, and sensitive - which is
exactly what is needed in BET assays.
to its absorption of light. " 3
Due to the nature of the microfluidics,
including the fluidic pathway and sample/lysate chamber, the Eclipse
only requires 1mL of lysate and ensures a 1:1 ratio of sample to lysate
for each assay.
Ensuring equivalent performance of the
Sievers Eclipse to traditional microplate
readers
Extensive testing was performed throughout the development of
the Eclipse to ensure equivalent performance to a 96-well microplate
reader. The United States Pharmacopeia outlines in chapter <1225>
the parameters to look at when validating an analytical method.
The performance of the Eclipse system was evaluated by measuring
different parameters outlined in USP <1225>.
Validation Parameter
Accuracy
Precision
Linearity
Limit of Detection (LOD)
Upper Range
Specificity
System Suitability
Equivalency to 96-well plate
Well Uniformity
Background Contamination
Table 1. USP <1225> Criteria and Sievers Eclipse
Sievers Eclipse
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1:1 ratio verification (robustness) 
The Eclipse microplate has a standard curve embedded in five
segments, and each standard has its own negative control. There are
21 sample segments that allow a user to run up to 21 samples on
one plate, similar to a 96-well microplate reader. Each of the sample
segments contain endotoxin for the Positive Product Control.
Because microfluidics requires smaller reaction volumes but does
not alter the biochemistry, the Eclipse microfluidic technology is
designed to use less lysate than typical methods, while maintaining
full compliance. The optical density for the Eclipse was calculated
using Beer's Law. Beer's Law, also known as Beer-Lambert's Law, states
that " the concentration of a chemical solution is directly proportional
59
American Pharmaceutical Review | May/June 2023
Testing was performed with multiple LAL lots from multiple vendors
over multiple years. In addition to LAL Reagent Water, drug product
samples were run on both the Eclipse and a 96-well microplate
reader. Data showed that the performance of the Eclipse was
equivalent to the traditional microplate reader. In conclusion, the
Eclipse uses a compendial kinetic chromogenic lysate to perform
a compliant Bacterial Endotoxin Test. Validation data according
to USP <1225> shows that the Eclipse is equivalent to the 96-well
microplate reader.
References
1.
2.
3.
Sievers Analytical Instruments. Determining equivalent onset optical density (OD) values
on the Sievers Eclipse BET platform.
McCullough, Karen Zink. The Bacterial Endotoxins Test: A Practical Approach. DHI
Publishing, LLC, 2011.
Helmenstine, Anne Marie. Beer's Law Definition and Equation. ThoughtCo, 2019. https://
www.thoughtco.com/beers-law-definition-and-equation-608172
https://www.thoughtco.com/beers-law-definition-and-equation-608172
APR May/June 2023

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