APR January/February 2022 - 58

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Table 1C. POC: Spiked Endotoxin Recovery Over Time
Evaluated endotoxin recoveries from spiked RSE in biologic drug product using rFC reagents for masking during storage
* Determined non-inhibitory dilution of biologic drug product using rFC.
* Spike drug product and LAL Reagent Water control to concentration of 2 EU/mL with RSE.
* Test using rFC reagents from Vendor A at Time 0. Store spiked sample at 20-25°C. Repeat test using rFC at Days 1 and 5.
Time Points
(Days)
Dilution
Spiked 2 EU/mL RSE Drug Product
(T0)
1
5
Spiked 2 EU/mL RSE Water Control
(T0)
1
5
100
100
Sample EU/mL
1.88
2.95
2.74
2.05
1.96
1.85
Sample Spike Recovery (%)
94
148
137
103
98
93
0.567
0.652
0.702
0.467
0.441
0.478
PPC Reserved
EU/mL
% PPC
110
125
135
89
84
92
Spiked endotoxin recoveries calculated against the theoretical spike of 2 EU/mL and corresponding %PPC met expected spike recovery of
50-200% at T0, T1, and T5 in the drug product and water control.
Risk Assessment
Category
A validation risk assessment was performed by ranking identified gaps
according to low, medium, or high risk. Identified gaps not covered by
published data, gaps that cannot be justified, or any gap with greater
severity than low were assessed during the method validation. The risk
assessment identified over 40 potential risk factors related to the rFC
method use. Risks were classified in seven categories and are listed in
Table 2.
No gaps were identified for most categories due to the available data
and detection controls in place. Many risks were considered low due
to their detectability during the method suitability and assay controls
performed for each assay and incoming test materials.
The reagent hold time is an example of sample and reagent handling
risk. There was no data or previous experience to support a hold time
of opened reagents, resulting in a medium ranked gap. Therefore, an
rFC reagent hold time study was carried out as part of the general
method validation to generate data and experience with the particular
reagent hold time conditions.
Roche General
Product-Independent Validation
At Roche's pilot laboratory, the objective of the validation strategy is
the implementation of rFC as the primary bacterial endotoxin method
while the LAL method would serve as a contingency method for the
testing of water samples. This Roche site uses the water to manufacture
products that are globally licensed. Therefore, the test for endotoxins
must meet the requirements for multiple pharmacopoeias.
Alternative methods to those listed in USP<85> for the detection
of endotoxins must be validated per USP <1223> Alternative
Microbiological Methods and <1225> Validation of Compendial
Procedures, in accordance to USP<1085>. The 2012 FDA Guidance
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Sample & reagent
volume
rFC Enzyme performance
Table 2. General Validation Risk Assessment
Example of potential
risk identified
Concentration of rFC Enzyme is
not optimal
rFC Enzyme cannot be activated
by endotoxins
Sample & reagent
handling
Reagents (fluorescence
substrate, rFC enzyme, buffer)
do not perform as expected after
opening, storage at 2 - 8°C and
mixing with freshly opened vials
Additional reagents used (e.g.
Tris-buffer, Pyrosperse) are not
compatible with rFC
Fluorescence & standard
curve
Result precision
Result accuracy
Sensitivity setting is not correct
(varies over time)
The plate reader affects
obtained results
False negative/positive results
are obtained/recovery of spiked
endotoxin is below 50% or
above 200%
Response to different endotoxin
sources
Response to beta glucans
Other
Single Source-Supply of rFC
Reagents
Risk reduction
measure
No gap identified
No gap identified
Open rFC reagent hold time
test
Compatibility check with
Tris-buffer
No gap identified
No gap identified
No gap identified
No gap identified
No gap identified
Additional supplier to be
validated in future. Traditional
Bacterial Endotoxin Test using
LAL-reagent will be used as
backup.
for industry on Pyrogen and Endotoxins Testing allows the use of an
alternative assay that provides advantages to those in the USP. This
guidance further states that such alternative methods should be
validated as described in USP<1225>.
General product-independent validation addresses parameters
robustness, specificity, linearity, range, the limit of quantitation,
accuracy, precision, and equivalence. Table 3 lists the validation
parameters and the evaluation approaches.

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