The document discusses an animal-free alternative for endotoxin testing using recombinant factor C (RFC) assays, which can detect bacterial endotoxins that cause severe health risks. RFC assays provide advantages such as improved sensitivity, reproducibility, and lower costs compared to traditional Limulus Amebocyte Lysate (LAL) tests. The text highlights the importance of endotoxin testing in various industries and regulatory requirements, emphasizing the need for methods that adhere to ethical standards by reducing animal use.

1. An Animal Free Alternative to Endotoxin Testing
Recombinant
Factor C
Assay
It can cause inflammation, fever, septic shock,
organ failure or even death.
They can easily contaminate samples and
products.
Endotoxins can survive sterilisation process.
Endotoxins testing is part of the regulatory
requirement for validation.
What is Recombinant
Factor C?
Recombinant Factor
C (rFC) is a
recombinantly
manufactured protein
used for the
detection of bacterial
endotoxins
It strongly binds to the LPS
and detect their presenceWhat is endotoxin?
Type of pyrogen
Present in cell wall of gram negative bacteria
Lipid A is responsible for the endotoxin activity
Why test for
endotoxins?
Endotoxins Limits
0.2 EU/kg ( Intrathecal )
5.0 EU/kg ( Intravenous & intramuscular )
ICH
Q4B annex 14
(Bacterial
Endotoxins Test
GeneralChapter)
Pharmacopoei
a
(Ph. Eur.), (JP)
& (USP)
Parenteral pharmaceutical
products
Medical
devices
Water for
injection
What is it tested on?
Control bioburden in the food
industry
Raw materials
Procedure
Binding
Step 1
Step 2
Wash
Detection
Step 3
90 minutes
37 degrees Celsius
Shaking
Repeat three times
90 minutes
37 degrees Celsius
Fluorescence
EndoLISA is new method for
demonstration of endotoxin
(LPS) levels where high affinity
binding of endotoxin by LPS-
specific phage protein is
followed by endpoint
fluorescence measurement
This assay enables the user to precisely determine the endotoxin level
192 tests, all reagents needed for running the assay included in
the kit
EndoLISA plates, pre coated microtiter strips
Endotoxin-Free Water
Binding Buffer
Endotoxin Standard
Wash Buffer
Enzyme
Substrate
Assay Buffer
Cover Foils
Product Format
without matrix effects
without the need for dilution
with a very broad measurement range
Water Used in rFC Assays
Ultrapure Water Sterile Water For
Injection
Water For Injection
Reproducibility of the assay
Greater control
which results in
a product with
a greater lot-
to-lot
consistency
Displays
consistency
and more
reproducible
results
Less variable
recovery values
for positive
product control
EndoLISA Sensitivity
Is the rFC Assay Better than the
LAL-Based Assays?
Simple to use
Rapid
Various compendial test
methods available
Requires a small amount
of test sample
Overall inexpensive
method
Highly standardized
LAL test doesn't discriminate
between living and dead bacteria
High initial expenses
False positives possible
Maximum valid dilution differs
between test methods
Nanoparticles may interfere with
one or more formats of the standard
limulus amoebocyte lysate
Lot-to-lot variability
Advantages
Limitations
LAL-Based
Assay
Endotoxin specific
Sustainable resource
Sensitivity range from 0.001 to
500 EU/ml
Lot-to-lot reproducibility
Approved by FDA
Comparable to other
quantitative LAL methods
Higher tolerance for
chaotropic agents, organic
solvents and detergents
Not a compendial test
Expensive
Only one test method
available
rFC-Based
Assay
EU Directive 2010/63
demands
The 3R - Replacement,
Refinement and
Reduction of animals
used for scientific
purposes
Do you know?
Advantages
Limitations