APR January/February 2022 - 56

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MICROBIOLOGY
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Validation Strategy for New Recombinant
Factor C Users
Evelyn Der
Senior Scientist, Roche Genentech, QC Technology Innovation and Implementation Group,
South San Francisco, CA
Carmen Marin
QC Scientist Microbiology, F. Hoffmann La Roche Ltd, Kaiseraugst, Switzerland
Introduction
This paper presents Roche's validation strategy of the recombinant
factor C (rFC) endotoxin testing method. The validation approach uses
statistical non-inferiority hypothesis testing based on spiked samples
with different concentrations of commercial endotoxin standard.
This approach is different from published literature studies while in
accordance with the current regulatory framework and streamlines
laboratory work. The validation activities are ongoing at the time
of this publication at a Roche pilot laboratory. As the validation
progresses, validation activities are to be continued at the other Roche
global network QC operations and applied for water and product
endotoxin testing.
Importance of a Sustainable
Alternative Method at Roche
Roche sponsors its corporate 3Rs Incentive Awards, aiming to
increase awareness and promote the dialogue among employees
and scientists about the 3-Rs for animal welfare (refine, reduce,
& replace). This program supports Roche's mission to support
innovation and advancement of animal welfare standards globally
and replaces non-sustainable animal dependent analytical methods
with alternative methods.
EU Directive 2010/63/EU adopted in 2010 by the Council of European
Convention, replaced an earlier Directive from 2018 and harmonized
standards across the EU. Based on the 3-R principles, this directive
increases awareness of animal protection and welfare. Its ultimate goal
is to replace animal use for scientific purposes (e.g., applied research,
regulatory testing and production, education, and training).
At present, Roche tests globally over 39,000 water samples annually
for endotoxins to monitor the quality of the water systems. Testing is
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Viviane Grunert da Fonseca, PhD
Non-clinical Statistician, Roche, MSAT (Manufacturing, Science and Technology),
Penzberg, Germany
Lindsey Silva, PhD
QC Director, Roche Genentech, Microbiology, South San Francisco, CA
dependent upon the compendia-based Limulus Amebocyte Lysate
(LAL) test. There would be a significant impact on horseshoe crab
protection using an alternative method.
Proof of Concept (POC) Studies
Prior to the pilot phase of the rFC project, Roche performed preliminary
proof of concept studies.
POC: Vendor Reagent Comparison
Different rFC vendor kits and respective readers were compared to
a LAL kit and reader, and analyst to analyst variability was assessed.
Expected recovery was 50 to 200% for the spiked Reference Standard
Endotoxin (RSE) recovery to rule out endotoxin inhibition and
enhancement. Endotoxin recoveries of the spiked samples were
compared against a theoretical spike of 5 EU/mL. For monoclonal
antibody (mAB) #1, a lower than expected 47% RSE recovery observed
with a vendor rFC kit and plate reader was likely equipment related.
The Positive Product Control (PPC) and spiked RSE recovery results
from the same and remaining vendors were acceptable, indicating the
potential suitability of the rFC method. Refer to Table 1A.
POC: Interchange of rFC Reagents with Different Plate Readers
Evaluated was the interchange of different vendor rFC reagents and
plate readers. Similar to the previous POC study, lower than expected
spiked RSE recoveries using Vendor C's plate reader and Vendor B's
reagents were attained. This event similarly was likely due to the initial
reader setup. The remaining results were acceptable for %PPC and
spiked RSE recoveries. Refer to Table 1B.
POC: Spiked Endotoxin Recovery Over Time
A mAB and water control were spiked with 2 EU/mL RSE to evaluate
possible storage-related masking effects.
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