APR January/February 2022 - 57

« MICROBIOLOGY
The spiked test sample and control were held at 20-25˚C and tested
at Time 0, Day 1, and Day 5. A comparison was made between the
endotoxin recoveries from the test and control samples at each time
point. Endotoxin masking was not evident. The data indicates that
the rFC method is suitable for bacterial endotoxin testing in a drug
product. Refer to Table 1C.
Regulatory Considerations
In Europe and as of January 2021, the rFC method is now an alternative
pharmacopeial method, Ph Eur 2.6.32. For the bacterial endotoxins test,
the monographs still reference Ph Eur chapter 2.6.14. Therefore, method
2.6.32 is considered an alternative to method 2.6.14 as described in
the General Notices. Replacing a prescribed monograph method with
another described in the Ph Eur does not require re-validation per
se, other than in consideration of its use for a specific substance or
product.16
To replace the Ph Eur method 2.6.14 with 2.6.32, a reference
to 2.6.32 should be added to the scope of these monographs as a
supplemental or replacement bacterial endotoxin method.
Outside of Europe, the rFC method is an alternative nonpharmacopoeia
method. In the US, rFC is considered an alternative
test in current USP <1085> since it is not a method in USP <85>. A
2019 USP<85> draft added the rFC method; however, this rFC addition
was cancelled, resulting in a draft standalone chapter <1085.1>.
Therefore, the rFC remains an alternative method in the USP.
The rFC method is considered an alternative method in the ChP 9251
(informational) and the 18th edition of the JP, recently published.
Some FDA Office of Pharmaceutical Quality (OPQ) reviewers were
either reluctant to accept BP/EP/JP quality standards or informed
companies that USP/NF standards must be used as specifications even
though the alternative standards are appropriate. The FDA's Office
of Pharmaceutical Quality MAPP (Manual of Policies and Procedure)
5310.7 clarifies for reviewers the appropriate use of quality standards
found in alternative compendia. MAPP 5310.7 offers NDA/IND/ANDA
sponsors the option of using standards from the alternative compendia
provided that the rigor and acceptance criteria are equivalent to or
better than USP/NF monograph requirements and the alternative
analytical procedures are based on similar principles and performance
characteristics (e.g., specificity, accuracy, precision).17
Table 1A. POC: Recommended rFC Vendor Reagents
Evaluated endotoxin recovery results with LAL KCA assay and rFC vendor kits with vendor recommended plate readers.
* RSE 4-Point Standard Curve (0.005 to 5.0 EU/mL)
* 0.5 EU/mL RSE PPC
* Two testers
Two Testers
(Tester 1/Tester 2)
Monocloral Antibody #1
Monocloral Antibody #2
Monocloral Antibody #3
LAL WATER
LAL KCA Vendor A
% Endotoxin Recovery2
72/55
102/93
96/86
120/144
% PPC2
91/93
97/113
90/98
79/113
Vendor A rFC Reagents
% Endotoxin Recovery2
83/61
135/130
138/104
129/149
% PPC2
150/135
171/153
156/149
116/134
Vendor B rFC Reagents
% Endotoxin Recovery2
59/471
82/82
86/65
100/100
1Expected 50 to 200% PPC and spiked endotoxin recoveries met except for reading performed with Vendor B reader. Likely related to equipment set-up.
2First reported value is from Tester 1. Second reported value is from Tester 2.
Acceptable standard curves, spiked RSE PPC recoveries of 50-200%, and coefficient of variation (≤ 25%; 


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APR January/February 2022

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