APR January/February 2022 - 59

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Attribute
Linearity
Range
Table 3. General rFC Method Validation (Performed at Roche)
Evaluation/Risk Assessment Required by References
These parameters were not evaluated
as separate attributes in the validation
because:
* Risk Assessment: Low risk based
on literature review
Limit of
Quantitation
* Standard curve is performed for
each plate
* Existing controls on incoming
reagents
Specificity
This parameter was not evaluated
separately in the validation because
this was assessed as low risk based
on a literature review (different
endotoxin sources & beta glucans and
reported endotoxin data for accuracy
and precision)
Robustness
Accuracy
Precision
Risk Assessment: Low to medium risks
Risk Assessment: Low risk based on
Literature Review. The evaluation for
all three attributes involved:
* Methods: rFC & LAL
* Sample Type: PW & WFI (endotoxin
free)
* Endotoxin Standard: RSE
Equivalence a
Data analysis: Statistical hypothesis
test (i.e. confidence interval used for
acceptance criteria)
FDA Guidance
2012 (& USP
<1223>)
Parameters bolded were evaluated as separate attributes in the validation.
a
2, 4, 5
USP <1225> &
<1223>
1, 3
1, 3, 9
1, 3
USP <1225> &
<1223>
3, 5, 7, 8, 11,
12, 14
and one sample of PW, both spiked with three RSE endotoxin levels
(0.01, 0.1, 1 EU/mL). In this way, the experimental setup generates
measurement variability representing within-laboratory variation
under routine operating conditions. Furthermore, each endotoxin
level is plated three times in duplicate on an rFC-assay plate and
three times in duplicate on a LAL-assay plate. Per data set, per run and
concentration, three reportable measurements for the two methods,
rFC and LAL, are obtained. Overall, the two data sets contain some
statistical dependence structure, which is taken into account in the
statistical analysis (statistical model). No PPCs were added and freshly
made assay standard curves were used for each run. Refer to Figure 1
for an illustration of the experimental design.
USP <1225> &
<1223>
USP <1225> &
<1223>
USP <1225> &
<1223>
2, 3, 6, 9, 10, 11,
12, 13, 14
2, 3, 6, 9, 12,
13, 15
2, 3, 9, 10,
11, 12
Product Specific Validation
Not verified on its own but rather with respect to parameters accuracy and precision.
Robustness was evaluated for risk conditions that were ranked higher
than low. Parameters ranked low in the risk assessment (i.e., specificity,
linearity, range, the limit of quantitation, accuracy, and precision that
are supported by published data) were not considered necessary to be
evaluated separately in the general validation.
Nevertheless, equivalency cannot be studied independently and is
considered not sufficiently justified due to the lack of statistically
representative data. Therefore, equivalency to LAL is evaluated
by the statistical hypothesis test for non-inferiority with respect
to accuracy and precision using relevant pharmaceutical water
samples spiked with low, medium, and high concentrations of RSE.
This demonstration of equivalency is adequate to limit the risk to
patient safety and is suitable for demonstrating specificity according
to USP<1225>.
The general method validation does not take into account samples
containing autochthonous endotoxin (i.e., non-standardized naturally
occurring) with comparability parameters. Instead, samples used are
spiked with USP RSE. The comparability of the rFC and LAL methods
with respect to different endotoxin sources (i.e., natural occurring
endotoxins) is supported with literature reviews.
The parameters equivalency, accuracy, and precision are assessed
experimentally using two data sets generated in the laboratory, one
data set with water for injection (WFI) samples and one data set with
purified water (PW) samples. Each data set is generated in twelve
independent runs performed by two different analysts on different
days, where each analytical run is associated with one sample of WFI
The minimum requirement of a product-specific validation for
rFC testing is aimed at product samples (DP, DS and IPCs). Productspecific
validation is the inhibition and enhancement test or what is
called method suitability. Testing is performed on three batches of
each sample type using rFC and LAL. This is required and performed
per USP<85>, EP 2.6.14, 5.1.10, and 2.6.32. PPCs are included with
each batch of sample type tested. Beta-glucan blocking buffer is not
necessary to overcome any possible interference with the rFC method
as this reagent does not include Factor G that would cause falsepositive
results. Treatment with a dispersant is not compatible with
rFC measurements due to fluorescence interference.
Statistical Analysis of ProductIndependent
Validation
The statistical analysis aims to demonstrate statistically significant
non-inferiority, of the rFC method compared to the LAL method, with
respect to accuracy and precision.
Why non-inferiority?
Equivalence, as a validation parameter, is seen in terms of assessing
the safety of an article (USP<1223>). As illustrated in Figure 2, there
is concern about the patient's safety in a situation of " inferiority " . On
the other hand, there should be no such concern under a situation of
" non-inferiority " , when rFC performs slightly worse, equal to or better
than the LAL-method, since a (much) more sensitive (Figure 2(a)) or
precise (Figure 2(b)) rFC performance, but also a slightly worse rFC
performance, does not compromise patient's safety.
A statistical non-inferiority hypothesis test, which decides between
the two situations of " inferiority " (H0
(H1 hypothesis), can limit the risk (to patient safety) of falsely deciding
in " non-inferiority " to a very low probability (usually 5%). It is therefore
a useful tool to verify the validation parameter " equivalence " according
to USP<1223>.
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| 59
hypothesis) and " non-inferiority "
»
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