COMMENTARY

Acceptable Levels of Endotoxin in Vaccine Formulations During
Preclinical Research

LUIS A. BRITO, MANMOHAN SINGH

Novartis Vaccines and Diagnostics, 350 Massachusetts Ave., Cambridge, Massachusetts 02139

Received 26 March 2010; revised 7 May 2010; accepted 10 May 2010

Published online 23 June 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22267

ABSTRACT: This brief commentary reviews endotoxin levels of commercial vaccines and puts
them into context for the preclinical researcher working in vaccines. Vaccines are not required to
adhere to endotoxin levels as outlined in the United States Pharmacopoeia. Vaccine manufac-
turers have to show that the vaccine is safe and efficacious in clinical trials. Endotoxin limits are
typically lot release specifications for most vaccines, but these values are not available to most
researchers designing preclinical experiments. The limits outlined are calculated from endo-
toxin levels found in a variety of vaccine types such as gene vectors, recombinant subunits,
polysaccharide, live attenuated, inactivated and toxoid vaccines. It is clear that certain families
of vaccines such as toxoids contain much higher levels of endotoxin, where others such as
purified recombinant subunits and gene vectors may contain very low levels.

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2010 Wiley-Liss,

Inc. and the American Pharmacists Association J Pharm Sci 100:34–37, 2011

Keywords:

endotoxin; vaccines; formulation; excipients; injectables; preformulation

INTRODUCTION

Researchers involved in preclinical vaccine research
typically have very little guidance on acceptable
endotoxin levels for preclinical evaluation. Previously
we defined endotoxin levels for preclinical evaluation
by using formulas released by the USP and calculat-
ing maximal endotoxin levels based on animal weight
using the USP recomended endotoxin limit of 5 USP-
EU/kg.

1

While this is technically correct for most

drugs, CFR610.13 states that, ‘‘the test for pyrogenic
substances is not required for the following products:
Products containing formed blood elements; Cryo-
precipitate; Plasma; Source Plasma; Normal Horse
Serum; bacterial, viral, and rickettsial vaccines and
antigens; toxoids; toxins; allergenic extracts; venoms;
diagnostic substances and trivalent organic arseni-
cals.’’

2

This update to our previous publication will

focus on reviewing the literature for endotoxin levels
found in commercial vaccines to allow the preclinical
researcher involved in vaccine development to set
appropriate endotoxin levels for the type of vaccine
they are developing such as a recombinant subunit

vaccine, a live attenuated viral vaccine, or a
polysaccharide conjugate.

ENDOTOXIN TESTING

There are a variety of tests available to measure
endotoxin levels, the gel clot technique, chromogenic
and end point turbidimetric or kinetic turbidimetric
techniques each with its own advantages or dis-
advantages. The gel clot technique allows for testing
of a very wide range of samples including samples
that are opaque such as emulsions, nanoparticle and
microparticle suspensions and alum; these samples
are unable to be tested with standard turbidimetric or
chromogenic techniques. The limitation of this assay
is that it is labor intensive and only a small number of
samples can be analyzed at once, without automation.
Chromogenic and turbidimetric assays can be run in a
96-well format. This allows for a higher throughput of
samples but are limited to samples that are clear
solutions. There are now units made specifically to
measure endotoxin levels such as Endosafe

1

by

Charles River Labs. This unit facilitates rapid testing
of samples with minimal user interaction, though it is
based on a turbidimetric system, so it does have
limitations on the types of samples that can be tested
on it.

Correspondence to: Manmohan Singh (Telephone: 617-871-

8100; Fax: [phone]; E-mail: [e-mail])

Journal of Pharmaceutical Sciences, Vol. 100, 34–37 (2011)
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2010 Wiley-Liss, Inc. and the American Pharmacists Association

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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 1, JANUARY 2011

ENDOTOXIN IN THE CLINIC

Endotoxin has been characterized as an agonist of
TLR 4. It is well known that even a small amount
(4 ng/kg) of endotoxin can cause a change in
physiology in humans.

3

Reports of adverse events

with endotoxin exposure have been well documen-
ted.

4

These findings justify the recommendations on

the amount of endotoxin in finished products passed
by the United States Pharmacopeia (USP), United
States Food and Drug Administration (USFDA), and
the European Medicines Agency (EMEA), in addition
to other regulatory agencies throughout the world.
Endotoxin values are listed as entotoxin units (EU)
since there is heterogeneity in the molecular weight of
endotoxin (it is not a single component).

There are few cases of endotoxin contamination in

parenteral products that have been administered to
patients.

5

One such example occurred when genta-

micin that was contaminated with endotoxin and a
nonendotoxin contaminate known to potentate an
endotoxin response. There were 210 adverse events
filed. The lots were tested by the FDA and 10% of
the lots tested showed elevated endotoxin levels
(above 5 EU/kg).

6,7

This event highlights that syner-

gistic contaminates can potentate the effect of
endotoxin.

5

A major reason for changing from a whole cell

pertussis vaccine to an acceular vaccine was to
decrease the reactogenicity of the vaccine.

8,9

The

high levels of endotoxin may have been one of the
causes of the reactogenicity, but this is not able to be
confirmed due to the over 3000 different components
in the whole cell vaccine.

10

This example highlights

how difficult it is for certain vaccines to distinguish a
response due to endotoxin or a response due to
another component or excipient in the vaccine.

ENDOTOXIN LEVELS IN COMMERCIAL
VACCINES

Vaccine manufacturers do not release their internal
lot release specifications on endotoxin levels of their
finished product. There are reports in the literature of
independent laboratories analyzing endotoxin levels
for a variety of vaccines. Table 1 outlines the findings
from the literature for a number of different
commercial vaccines. These values should not be
taken as limits set by companies, but only what has
been detected in a series of vaccines analyzed
throughout the years.

These levels are noted as EU/mL. As mentioned

previously the USP sets endotoxin limits by EU/kg.
Given the average volume of 0.5 mL per human
vaccine dose the values can be back calculated to
compare them to the USP limits. Since vaccines are
not bound by the endotoxin limits stated in the USP
we are recommending the following endotoxin level
recommendation for various vaccine types summar-
ized in Table 2 based on our findings from the
literature. A recommendation of <10 EU/mL for
genetic vectors was chosen due to the extensive
purification needed for these types of vaccines. A level
of <20 EU/mL for recombinant subunits and poly-
saccharide vaccines was recommended due to the
high purity and highly controlled method of manu-
facture for these vaccine types. A recommendation of
<200 EU/mL for live attenuated vaccines is based on
the complex nature of these vaccines containing
multiple antigens. A similar rational goes into the
recommendation of <500 EU/mL for inactivated
vaccines. Toxoid vaccines have the highest recom-
mended values of <200,000 EU/mL due to the
complexity of these vaccines and the fact that
they are all derived from bacteria with minimal

Table 1. Reported endotoxin levels for various vaccines

Vaccine

Amount of Endotoxin

References

DTaP

Less than 100 EU per dose, 0.288–1390.8 EU/mL

9,11

DTwP

7600–181,640 EU/mL

9,12

Influenza

10–100 ng/mL, 0.304–380 EU/mL

12,13

HIB

<1.2–<12 EU/dose, 10.9–220.4 EU/dose

14,15

Prevnar

<0.21 EU/dose

16

Small Pox

Undetected

12

Rubeola

Undetected

12

Rubella

10 EU/mL

12

Polio

Undetected—100 EU/mL

12

Rabies

100 EU/mL

12

Mumps

100 EU/mL

12

Typhus

100,000 EU/mL

12

Cholera

1,000,000 EU/mL

12

Viral/nonviral vector

<10 EU/mL

17,18

1 ng of endotoxin

¼ 10 EU. This is a general comment that allows readers to convert the levels in the table to ng

of endotoxin based on reference 1. There is no specific link within the table.

DOI 10.1002/jps

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VACCINE FORMULATIONS DURING PRECLINICAL RESEARCH

35

purification. Though advances in purification and
process have been continuously improving the
endotoxin levels in these types of vaccines. We are
proposing these values as guidelines for evaluating
antigens in all animal studies during early develop-
ment. As vaccine development progresses to the
clinic, safety and efficacy will be the ultimate readout.
Higher endotoxin levels than outlined in Table 2 may
be acceptable in certain instances, though research-
ers should ensure they are adequately addressing
endotoxin levels.

The FDA has also released guidelines on how

combination vaccines should be developed and
tested.

19

The guidance states that ‘‘Each bulk

component of a combined vaccine should meet purity
characteristics appropriate for that component. For
example, the Haemophilus b conjugate component
should meet the acceptance criteria for pyrogenicity
testing even if combined with a vaccine such as DTP
which is known to contain endotoxin. In this case,
final product will not be pyrogen tested.’’

19

This

further highlights that endotoxin levels are specific to
each licensed vaccine and a general statement as
what is found in the USP guidance for endotoxin do
not apply. It should be noted that a pyrogen is a
substance endogenous or exogenous that induces a
febrile response, endotoxin is only one type of
pyrogen.

20

Pyrogenicity testing looks at the febrile

response typically in rabbits, and does not look at the
specific cause of the increased temperature.

5

CONCLUSION

During preclinical development of the next genera-
tion vaccines endotoxin levels should be closely
monitored despite the heterogeneity in licensed
vaccines. Routine measurement of endotoxin levels
is necessary to properly understand preclinical
animal studies results. Importantly high endotoxin
levels in a preclinical setting should not preclude its
use for immunogenicity studies assuming all reason-

able steps have been performed to try and remove any
residual endotoxin. However as the purification
process gets further optimized, the residual endotoxin
levels may drop off and the subsequent immunogeni-
city data with the same dose of antigen may show a
reduction. The levels outlined above should only act
as a starting point for researchers. Ideally endotoxin
levels should be as low as possible to allow for an
appropriate evaluation of the antigens being tested.
With the evaluation of adjuvants, it is even more
important to dissect the response of the adjuvant by
having a low residual endotoxin coming from the
purified antigen. The endotoxin levels should also be
closely monitored due to the potential of a synergistic
effect.

REFERENCES

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Table 2. Recommended endotoxin levels for different
vaccine types

Vaccine Type

Maximum Recommended

Endotoxin Levels (EU/mL)

Gene vector

<10

Recombinant subunit

<20

Polysacharide

<20

Live attenuated

<200

Inactivated

<500

Toxoid

a

<200,000

a

Based on values from DTwP, and Cholera vaccines, through purification

and process changes endotoxin levels should fall below 2000 EU/dose (based
on DTaP).

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BRITO AND SINGH

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