ASSIGNMENT ON

Different types of Endotoxin and Exotoxin

Course name: Pharmacy Quality Assurance
Course code: PHRM 405


SUBMITTED TO:
Dr. Shamsun Nahar Khan
Associate Professor
Department of Pharmacy
East West University


SUBMITTED BY:
Samiya Khondaker Rinta
ID: 2010-3-70-048




Submission date: 19
th
February, 2014


BACTERIAL TOXIN: EXOTOXIN AND ENDOTOXIN
Bacterial toxin is a poisonous substance, especially protein in nature, which can be produced by
both gram positive and gram negative bacteria and is capable of causing disease when introduced
into the human body. Bacterial toxins are exotoxins and endotoxins (Hayat, 2013). Bacterial
toxins can also be termed as bacterial pyrogen. A pyrogen is defined as any substance that can
cause a fever.

Figure 1: About endotoxin and exotoxin (Midlands Technical College, 2012).
Characteristics and differences of exotoxins and endotoxin are listed below:
Characteristics Exotoxins Endotoxins
Source Living gram positive and gram
negative bacteria
Lysed gram negative bacteria
Location Released from the cell Part of cell
Chemical composition Protein Lipopolysaccaride
Heat sensitivity Heat sensitive and inactivated at
60-80C. Toxicity destroyed over
60C.
Heat stable to 250C. Toxicity is
not destroyed above 60C for
hours.
Immune reactions Strong Weak
Conversion to toxoids Possible Not possible
Fever Usually do not produce fever in
the host.
Usually produce fever in the host
by release of interleukin-1 and
other mediators.
Specificity Specific to particular bacterial
strain
Non specific
Antigencity Highly antigenic. Stimulate
formation of antitoxin. Antitoxin
neutralizes the toxin.
Weakly antigenic. Antibodies
are protective.
Examples Streptococcus Salmonella typhi
Table: Differences between exotoxins and endotoxin (Hayat, 2013)
TYPES OF ENDOTOXIN AND EXOTOXIN
Types of endotoxin are:
1. Lipopolysaccharide (LPS): It is a long chain endotoxin; the most common form in Gram-
negative bacteria.
2. Lipid A: It is the lipid component of LPS. This lipid portion of endotoxin that is responsible
for its toxicity by binding to LBP and inducing the inflammatory cascade.
3. Lipooligosaccharide (LOS): It is a short chain endotoxin; found in N. meningitidis bacteria
that cause meningococcemia (Midlands Technical College, 2012).
The basic types of exotoxin based on structure and function:
1. A-B toxins
2. Membrane disrupting toxins
3. Superantigens
4. Representative Exotoxins- Cytotoxins, Neurotoxins and Enterotoxins (Midlands Technical
College, 2012).
GENERAL CHEMICAL STRUCTURE
Most of the work on the chemical structure of endotoxin has been done with species of
Salmonella and E. coli. Lipopolysaccharides are complex amphiphilic molecules with a mw of
about 10kDa, that vary widely in chemical composition both between and among bacterial
species. The general sttructure of LPS is shown below.

Figure 2: General architecture of Lipopolysaccharide (Todar, 2012).

Figure 3: General Structure of Salmonella LPS. Glc = glucose; GlcNac = N-acetyl-
glucosamine; Gal = galactose; Hep = heptose; P = phosphate; Etn = ethanolamine; R1 and R2
= phoshoethanolamine or aminoarabinose. Ra to Re indicate incomplete forms of LPS. The
Rd2 phenotype (not shown) would have only a single heptose unit. The Rc, Rd2, and Rd1
mutants lack the phosphate group attached to Hep (Todar, 2012).

Figure 4: Complete structure of the Lipid A moiety of LPS of S. typhimurium, S. minnesota,
and E. coli (Todar, 2012).
EXAMPLES OF BACTERIA COMMONLY SUSCEPTIBLE TO GROW IN
PHARMACEUTICAL INSTRUMENTS AND PREPARATIONS
Good examples of endotoxin producing gram-negative bacteria are Escherichia coli,
Pseudomonas aeruginosa and Enterobacter (Silverman and Ostro, 1999).
Examples of exotoxin producing bacteria are Escherichia coli (produces enterotoxins),
Streptococcus pyogenes (produces superantigens), Staphylococcus aureus (produces A-B toxins)
(Midlands Technical College, 2012).
DEPYROGENATION PROCESSES IN PHARMACEUTICALS
1. Ion exchange chromatography: Endotoxins are negatively charged, and will bind to an
anion exchanger. If the target substance is not also negatively charged, it will pass through
the column before the endotoxin, and an effective separation can be achieved. Typical
examples of endotoxin binding ligands include histamine, nitrogen-containing heterocyclic
compounds, and polymyxin B.
2. Cation exchange chromatography: An alternative to anion exchange is cation exchange
chromatography, in which positively charged solutes bind to the solid chromatographic
media. In this method, the target binds to the column instead of the endotoxin. The
endotoxin then washes through the column, and a pure target is later eluted off the column.
3. Ultrafiltration: Because the molecular weight of endotoxins is usually over 10 kD,
ultrafiltration can sometimes be used to perform as a size based separation. This method is
best used only when all endotoxins present are larger than 300,000 Da (U.S. FDA, 2009).
4. Distillation: This method is also based on the large molecular weight and heat stability of
endotoxins. Low molecular-weight solvents can be easily purified by boiling and collecting
the condensed vapor in an endotoxin free vessel. The large LPS molecules do not easily
vaporize, and are thus left behind in the heating vessel (U.S. FDA, 2009).
5. Heating: Heating methods, especially dry heat methods are most commonly used to sterilize
and /or depyrogenate pharmaceutical glass wares and other lab equipments. Heat is applied
by baking in a dry heat oven that is designed specifically for the depyrogenation process.
Although endotoxins are relatively thermally stable, sufficient heating (250C for 30 min)
results in a 3log reduction of endotoxin levels (Agalloco and Frederick, 2008).
PROCESSES USED IN PHARMACEUTICALS FOR INACTIVATION/DESTRUCTION
OF PYROGENS
1. Acid-base hydrolysis: This method has been shown to cleave Lipid A from the
polysaccharide in the LPS molecule. The lipid moiety alone is not soluble in water. Thus
unable to bind to endothelial cells, it is rendered inactive. However, acid-base hydrolysis
can denature a target protein, and is thus unsuitable when purifying a protein.
2. Oxidation: Oxidation using hydrogen peroxide or ethylene oxide is often used as a
pyrogen destroying solution. The mechanism for this destruction is unknown (U.S. FDA,
2009).
3. Sodium Hydroxide: When purifying proteins, sodium hydroxide (NaOH) can be used
safely and effectively. It is also widely used for depyrogenation of non-autoclavable
equipment (e.g. plastics) and chromatography columns. In fact, when using an anion
exchanger to remove pyrogens, it is necessary to clean the column with NaOH after each
batch.











REFERENCES
Agalloco, J.P. and Frederick, J. (2008) Validation of Pharmaceutical Processes. 3rd edition,
Informa Healthcare USA, New York.

Hayat, K. (2013) Difference between Endotoxin and Exotoxin Medimoon: Medical Available
from: http://medimoon.com/category/medical/ [Accessed 18th February 2014]

Midlands Technical College (2012) Microbial Mechanisms of Pathogenicity. Available from:
http://classes.midlandstech.edu/carterp/Courses/bio225/chap15/lecture3.htm [Accessed 18th
February 2014]
Silverman, M. H. and Ostro, M. J. (1999) Bacterial Endotoxin in Human Disease XOMA Ltd
Todar, K. (2012) Bacterial Endotoxin Textbook of Bacteriology. Available from:
http://textbookofbacteriology.net/endotoxin.html [Accessed 18th February 2014]
U.S. Food and Drug Administration (2009) Bacterial Endotoxins/Pyrogens. Available from:
http://www.fda.gov/ICECI/Inspections/InspectionGuides/InspectionTechnicalGuides/ucm07291
8.htm [Accessed 18th February 2014]