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Evidence Against a Bacterial Endotoxin Masking Effect in

Biologic Drug Products by Limulus Amebocyte Lysate
Detection
Jay S. Bolden, Mark E. Claerbout, Matthew K. Miner, et al.

PDA J Pharm Sci and Tech 2014, 68 472-477

Access the most recent version at doi:10.5731/pdajpst.2014.00999
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RESEARCH

Evidence Against a Bacterial Endotoxin Masking Effect in

Biologic Drug Products by Limulus Amebocyte Lysate
Detection
JAY S. BOLDEN*, MARK E. CLAERBOUT, MATTHEW K. MINER, MARIE A. MURPHY, KELLY R. SMITH,
and ROB E. WARBURTON

Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN ©PDA, Inc. 2014

ABSTRACT: The inability to detect endotoxin using compendia methods is a potential safety concern for patients due to
the lack of endotoxin removal capabilities at the fill–finish stage in typical aseptic biologic drug product manufacturing.
We have successfully demonstrated endotoxin challenge study recovery methodology using mammalian cell–produced
biologic drug products and drug substances in citrate, histidine, phosphate, and sodium acetate buffer formulations
containing polysorbate, challenged with an endotoxin analyte, for up to 6 months of storage. Successful recovery was
similarly demonstrated for a preserved, peptide-containing drug product formulation. To isolate a potential masking— or
low-endotoxin recovery—source, additional studies were performed to evaluate factors including product manufacturing
contact surfaces, drug product matrix with and without polysorbate, individual matrix components, protein concentration,
reagent suppliers, an orthogonal test method, and storage conditions. In all cases, acceptable recoveries were observed.
Bacterial endotoxin is known to be chemically stable at physiological conditions. Purified endotoxin in aqueous conditions
is likely to self-aggregate or bind to surfaces. Neither the nature of, nor the storage conditions of, the studied formulation
matrices were shown experimentally to render the challenge endotoxin biologically inactive. The results highlight the
importance of appropriate study design in assessing the recovery of endotoxins.

KEYWORDS: Bacterial endotoxins, Masking, Pyrogens, Chromogenic, Recombinant Factor C, Phosphate, Citrate,
Polysorbate.

LAY ABSTRACT: Bacterial endotoxin is a Gram-negative bacterial cell wall component that is harmful to humans at
threshold concentrations, and it is not expected to be in aseptically-produced pharmaceutical medicines. It has been
suggested that endotoxin cannot be detected over time in certain biopharmaceutical drug product formulations containing
citrate, phosphate, and polysorbate components via an unknown masking mechanism. We have generated and present data
here that indicate that endotoxin can be recovered in a variety of matrices, and under various experimental conditions.

Introduction sive microbial and pyrogen control/monitoring strategy

is required to assure consistent removal and control dur-
The control of pyrogens, but especially bacterial endo- ing the purification steps for the drug substance and
toxins, in the production of parenteral pharmaceutical formulated drug product. The primary pyrogen source is
products is one of many requirements to assure patient endotoxin from Gram-negative bacteria potentially pres-
safety. Bioproducts, especially those produced in mam- ent in the microbial flora of the manufacturing equipment
malian cell culture, have numerous manufacturing steps and aqueous matrices. The purification processes for
involving complex growth media and buffer matrices drug substances are designed to minimize adventitious
that promote microbial growth. Therefore a comprehen- microbial contamination, but they are not sterile. There-
fore bioburden and endotoxin monitoring of the in-pro-
cess stages of purification to the final drug substance, as
*Corresponding Author: Jay Bolden, Lilly Corporate well as the formulation of product at the aseptic fill–
Center, Indianapolis, IN 46285. Phone: ⫹1-317-276- finish steps, are routinely performed with results evalu-
9183; e-mail: bolden_ [email protected] ated against established limits. The techniques described
in United States Pharmacopoeia (USP) General Chapter
doi: 10.5731/pdajpst.2014.00999
⬍85⬎ (1), and harmonized with the European (2) and

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Japanese Pharmacopoeias (3), are typically employed (ATCC) 12014 was grown overnight in several tryp-
for endotoxin monitoring of these various matrices. Part ticase soy broth tubes. Individual tube contents were
of the compendia method verification suitability for each pooled into a polypropylene tube and then centrifuged
matrix type requires demonstration that the sample, as at 3200 relative centrifugal force for 10 min. The broth
diluted and prepared for testing, is within biological pH supernatant was decanted and the cell pellet resus-
range, and that a positive control derived from a control pended in Limulus amebocyte lysate (LAL) reagent-
standard endotoxin (CSE) can be recovered within 50 – grade water (LRW). The suspension was boiled in a
200% when applied to the test dilution. The CSE is a water bath at approximately 100 °C for 10 min. The
chemically purified lipopolysaccharide (LPS) prepara- contents were transferred to a polystyrene tube. Or-
tion derived from an Escherichia coli strain used to ganism kill was verified by analysis of a suspension
produce USP reference standard endotoxin (RSE). aliquot on a trypticase soy agar (TSA) pour plate. The
TSA plate was incubated at 30 –35 °C for not more
Endotoxin challenge studies are designed such that sam-
than 5 days with no growth observed. The stock sus-
ples are challenged with the analyte in the final sample
pension was assayed for endotoxin concentration by
container and tested initially and over various time in-
analyzing stock dilutions using the kinetic chromo-
tervals to evaluate endotoxin recovery. Other experimen-
genic method, and then stored at 2– 8 °C in the poly-
tal parameters, such as endotoxin source, challenge con-
styrene tube. Bowers and Tran (6) describe a NOE
centrations, and recovery criteria, are defined in the
preparation using filtration to remove the challenge
experimental design. The 2012 U.S. Food and Drug
organism for use in similar studies.
Administration guidance (4) recommends that protocols
designed to demonstrate stability of assayable endotox-
ins contents should consider the source of endotoxin Endotoxin Challenge Study Sample Preparation
used in these studies, as native endotoxins might react
differently than CSE. The guidance publication pre-dated The stock endotoxin concentration was quantified
the low endotoxin recovery (LER) issue, however, the prior to use in a challenge study by dilution of the
requirement to perform endotoxin challenge studies have stock onto 0.05 to 5.0 EU/mL or 0.01 to 1.0 EU/mL
been interpreted from the guidance. standard curves using CSE calibrated against the RSE.
Based on the observed value, the volume necessary to
The following experiments were designed and exe- challenge 0.05 to 0.1 mL of diluted stock to the study
cuted to understand the factors that may lead to re- matrix was calculated, such that after dilution to the
covery issues when challenging endotoxin directly to a non-interfering concentration in LRW as determined
sample solution, and then analyzing the sample over by the compendia inhibition/enhancement test, the
time using the compendia-verified test method. Public challenge concentration would be approximately the
presentations (5) have indicated that the LER phenom- mid-point of the chosen standard curve. The mid-point
enon is of specific concern for biologics in citrate and of the curve was chosen due to the increased variabil-
phosphate matrices containing polysorbate. ity created by the logarithmic reduction of the assay at
the high curve concentration, and because kinetic test-
The compendia photometric kinetic chromogenic as-
ing is not typically designed to be an endpoint limit
say was used to evaluate various drug products and
test. At each time point, the container was removed
components when challenged with a laboratory-prepared
from storage, vigorously vortex-mixed at least 5 min,
naturally occurring endotoxin (NOE) or LPS. We have
sampled, diluted to the test concentration, and ana-
demonstrated recovery in citrate and phosphate matrices
lyzed by the compendia method. We challenged single
containing polysorbate in addition to other biologic drug
containers that were amenable to opening multiple
product formulation matrices. The use of a NOE as a
times and containing adequate volume (i.e., drug prod-
model challenge analyte is superior to LPS both in per-
uct vials and drug substance sample tubes), whereas
formance and as a more representative analyte.
replicates were challenged for small-volume items
Materials and Methods such as semi-finished syringes. The containers were
challenged by removing the stopper, plunger or lid;
NOE Stock Endotoxin Preparation adding the challenge aliquot with a pipette; and re-
closing the container. All samples were stored at
To better simulate the natural analyte, an E.coli O55: 2– 8°C as is representative of the respective drug prod-
B5 stock from American Type Culture Collection uct and drug substance storage conditions.

Vol. 68, No. 5, September–October 2014 473

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LAL Detection Methodology

The kinetic chromogenic method described in the

compendia was used in combination with a Biotek
Elx808 absorbance reader measuring optical density
(OD) change at 405 nm and a 0.2 OD cutoff. Lonza
WinKQCL™ software was used for sample analysis.
Lonza LAL and CSE reagents were primarily used, but
Charles River Endosafe and Associates of Cape Cod
kinetic chromogenic reagents were also evaluated to
investigate a potential supplier effect on LER.
Figure 1
The Lonza Pyrogene® recombinant Factor C (rFC) sys-
tem was used as an orthogonal non-LAL detection meth- Y-intercept variability is inherent to the biological
odology. The rFC method is a 60 min fluorogenic end- nature of the assay. Endotoxin challenge recoveries
point test using a Biotek FLx800 instrument with 380/ were observed in three different batches of the same
440 nm excitation and emission wavelengths. Data was drug product (DP1, DP2, DP3) to shift with the inter-
analyzed using Lonza WinKQCL™ software. A three- assay y-intercept values. The average y-intercept vari-
part enzyme master mix reagent is used in combination ability was 0.75% across this data set and corresponded
with the same CSE used in the LAL methods, and to an average 75% recovery from the initial challenge.
unknown values are plotted against a positively-corre-
lated curve.
is drug product formulation without drug substance,
The acceptance criteria for linearity as described in the and water were challenged with a NOE. Product con-
compendia were used to determine assay and sample tact surfaces included stainless steel, polyvinylidene
validity, in addition to in-house y-intercept, slope, and difluoride (PVDF) filtration, polystyrene, silicone tub-
replicate percent coefficient of variance (%CV) sys- ing, glass, and bromobutyl rubber.
tem and data criteria. Recovery acceptance criteria
was chosen to be ⫾0.5 log from the initial time point The data from this study indicated that mixing is a critical
recovery. This criterion was chosen for (a) alignment recovery factor. Endotoxin was not recovered, or was re-
with compendia microbial challenge testing, and viral covered very poorly, during a gentle formulation mix with
challenge testing; others have used the compendia- a stir bar in the stainless steel container. Additional exper-
derived 50-200% positive control recovery criteria (5, iments were conducted to isolate the potential source of the
7); (b) a theoretical demonstration that a 1% y-inter- low recovery. Similar protein solutions were challenged
cept shift can cause a 36% recovery shift (8); (c) with endotoxin and held in stainless steel containers for 20
observed recovery shifts due to corresponding stan- days, in the final drug product container closure system for
dard curve y-intercept shift (Figure 1); and (d) the 6 months, and in polystyrene sample tubes for 60 days.
picogram-level detection capability of the assay. Figures 2a– e summarize the results of these studies. In
contrast to the initial experiment, the endotoxin challenge
Results and Discussion was successfully recovered in all cases. This result rein-
forces that rigorous mixing is essential to ensure the recov-
Citrate Drug Substance and Product Matrix ery of endotoxins when measured using the compendia test
Containing Polysorbate 80 method.

An initial study was conducted to address contact This same study design was executed in the drug
surface loss concerns in which drug product manufac- substance manufacturing site laboratory. The drug
turing was simulated from formulation compounding substance was challenged in the final polystyrene sam-
to pre-filled syringe filling. The target concentration ple container then stored at refrigerated (2– 8°C) and
was 20 EU/mL in the drug product, or 0.5 EU/mL after ultra-low temperatures (– 65 °C) for 14 days. The drug
dilution. This drug product contained relatively low substance is in a similar citrate and polysorbate 80
protein concentration (⬍5 mg/mL), 10 mM citrate and matrix as the drug product but contained approxi-
0.02% polysorbate 80. Drug product, placebo, which mately 40 mg/mL protein concentration. The results of

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Figure 2

(a) The initial formulation experiment resulted in poor recoveries. However, recovery was demonstrated in
subsequent studies, which indicates no recovery effect due to (b) the drug product and container closure, (c)
the formulation tanks, (d) the buffer, or (e) the drug product and polystyrene sample tubes.Two different
batches of the same drug product (DP) were used in the study (DP1, DP2).Placebo 1X in panel (d) is the nominal
concentration, which was subsequently diluted 2fold to a 1/8 concentration.

this experiment are shown in Figure 3 and indicate Histidine/Glycine Drug Substance and Product Matrix
there is no effect on recovery due to storage temper- Containing Polysorbate 80
ature or protein concentration.
A study was performed at a drug substance manufac-
To evaluate the effect of higher protein concentration, turing site laboratory; it simulated the penultimate
additional studies were conducted on formulation con- drug substance manufacturing step through to the drug
taining 80 –140 mg/mL protein in 20 mM citrate and product vial fill, using maximum production hold
0.02– 0.03% polysorbate 80 matrices. Another arm of times. The drug product and drug substance are in
this study evaluated Charles River Endosafe and As- similar matrices and contain approximate concentra-
sociate of Cape Cod kinetic chromogenic, and Lonza tions of 10 –15 mg/mL protein concentration, 140 mM
Pyrogene® rFC reagents. The endotoxin challenge histidine/glycine buffer, and 0.01% polysorbate 80.
was successfully recovered over 60 days using each
supplier’s LAL (Figure 4). The results indicate that The drug substance was challenged within a stainless
higher protein concentrations do not affect recovery steel container, held refrigerated for 24 h, then 0.22
and that there is no apparent LAL vendor-specific ␮m filtered into a polyethylene bag and stored refrig-
effect. Additionally, the capability of the Lonza Pyro- erated for a total of 14 days. The drug product was
gene® system as an orthogonal method to detect the challenged while in a polyethylene bag, held 48 h
endotoxin challenge was demonstrated. refrigerated, then 0.22 ␮m filtered, filled into vials,

Vol. 68, No. 5, September–October 2014 475

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Figure 3
Figure 5
Endotoxin challenges were recovered from both high
(>140 mg/mL, DS1) and moderate (⬃40 mg/mL, Endotoxin challenges were recovered from additional
DS2) protein concentration drug substances in citrate drug product (DP) matrices including phosphate, so-
and polysorbate 80 when stored at both refrigerated dium acetate and histidine/glycine matrices and a
and ultra-low temperatures, indicating no recovery ef- histidine/glycine drug substance (DS) matrix. Both
fects due to temperature or high protein concentrations. the NOE and LPS were used in the acetate and
phosphate studies, and both were recovered.

and stored refrigerated for a total of 14 days. Both the Additional Studies
drug product and drug substance were challenged with
Additional matrices were challenged in the final drug
endotoxin at 20 EU/mL, or 2.5 EU/mL after product
product container and held refrigerated. These matrices
dilution in LRW (Figure 5). The endotoxin challenge
included antibody drug products in a polysorbate matrix
was recovered in both the drug product and drug
buffered with either sodium phosphate or sodium acetate,
substance for up to 14 days of storage.
and peptides in metacresol-preserved glycerin at low pH,
or tris buffer at neutral pH matrices.

An arm of these studies also evaluated the NOE stock

challenge suspension against a commercially available
LPS. No practical difference was observed between the
endotoxin sources in the phosphate and acetate buffer
matrices. Valid, but low, recoveries were observed in the
preserved peptide glycerin at low-pH matrix when using
the NOE, and recoveries below acceptance criteria were
observed when using the LPS. To rule out the effect of
the preservative, a second peptide in a tris buffer matrix
at neutral pH was evaluated. The challenge was success-
fully recovered in the neutral pH peptide drug product
using either NOE or LPS, although the NOE was less
Figure 4 variable (Figure 6).

Endotoxin challenges were recovered from two The same endotoxin stock suspension preparation was
high-protein concentration drug product (⬃120 used repeatedly at the originating site laboratory, with
mg/mL) batches (DP1 and DP2) containing citrate a quantitation control performed prior to each experi-
and polysorbate 80 in the container closure system. ment. When stored under refrigeration as a liquid sus-
Recovery was demonstrated in DP2 using conven- pension in a polystyrene container, the stock averaged
tional LAL kinetic chromogenic methodology 4.2 ⫻ 106 EU/mL (%CV ⫽ 26%) over 398 days to date.
across three LAL suppliers, and using recombinant The observed variation is susceptible to the assay y-in-
Factor C by fluorogenic endpoint methodology. tercept variability. Y-intercept variability in endotoxin

476 PDA Journal of Pharmaceutical Science and Technology

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strains specific to a supplier’s CSE/LAL are publicly

available, can be used to maintain specificity to sup-
plier CSE, and fit the use of a model organism for
endotoxin challenge studies.

Acknowledgements

Dr. Dayue Chen, Dr. James Cooper, Dr. Michael

DeFelippis.

Conflict of Interest Declaration

Figure 6
The authors declare that they have no competing
A preserved peptide solution at approximately pH 4.0 interests.
(Pep1) was observed to have lost the commercial
endotoxin challenge in 4 days. Reduction to around References
the lower acceptance criteria was observed when us-
ing a NOE, indicating the purified LPS is more sus- 1. Chapter ⬍85⬎ Bacterial Endotoxins Test. In
ceptible to matrix pH affects. A similar preserved United States Pharmacopeia; United States Phar-
peptide buffered at pH 7.4 with Tris (Pep2) suitably macopeial Convention: Rockville, MD, 2013.
recovered both NOE and LPS challenges.
2. Chapter 2.6.14 Bacterial Endotoxins. In European
Pharmacopoeia; The European Directorate for
challenge studies could potentially be mitigated by ana- the Quality of Medicines & Health Care: Stras-
lyzing subsequent time points to the initial standard bourg, France, 2014.
curve. The choice to use the same E. coli strain as used
to prepare the internal platform commercial CSE is one 3. Chapter 4.01 Bacterial Endotoxins Test. In Japa-
of assured specificity, and for the same reasons that the nese Pharmacopoeia; Japanese Ministry of
RSE E. coli was chosen as a model endotoxin. Health, Labour and Welfare: Tokyo, Japan, 2011.
Conclusion
4. U.S. Department of Health and Human Services,
The NOE challenges were successfully recovered in Food and Drug Administration, Guidance for In-
the matrices of interest, and the LER effect could not dustry—Pyrogen and Endotoxin Testing: Ques-
be reproduced for citrate and phosphate buffer systems tions and Answers, June 2012.
containing polysorbate. Therefore, the respective ver-
ified test methods are capable of detecting potential 5. Chen, J. Low Endotoxin Recovery in Common
endotoxin in citrate, histidine/glycine, phosphate, and Biologic Products. Presented at PDA 8th Annual
sodium acetate matrices containing polysorbate, and are Global Conference on Pharmaceutical Microbiol-
not affected by time, typical protein concentration, re- ogy, Bethesda, MD, 2013.
frigerated or frozen storage conditions, process product-
contact materials, or LAL test reagent supplier. Recovery 6. Bowers, K.; Tran, L. Creation of an in-house
was also demonstrated using an orthogonal non-LAL naturally occurring endotoxin preparation for use
method. No evidence was found that indicates the LER in endotoxin spiking studies and LAL sample hold
phenomena posed a risk of failing to detect endotoxin time analysis. Am. Pharm. Rev. 2011, 14 (6).
during end-product testing by compendia methods.
Based on our findings, the harmonized compendia bac- 7. Williams, K. Endotoxin test concerns of biolog-
terial endotoxin chapters should continue to be used for ics. Am. Pharm. Rev. 2013, 16 (6).
routine endotoxins testing.
8. McCullough, K. The Bacterial Endotoxins Test: A
It is proposed here that a NOE liquid stock endotoxin Practical Guide. PDA and Davis Healthcare In-
preparation is as stable, representative, and suit- ternational Publishing, LLC: River Grove, IL,
able— or better—than an LPS. The E. coli endotoxin 2011; pp 48 – 49.

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