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ASSIGNMENT ON

Different types of Endotoxin and Exotoxin

Course name: Pharmacy Quality Assurance

Course code: PHRM 405

SUBMITTED TO:

Dr. Shamsun Nahar KhanAssociate Professor

Department of Pharmacy

East West University

SUBMITTED BY:

Samiya Khondaker Rinta

ID: 2010-3-70-048

Submission date: 19 th February, 2014

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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 introducedinto 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 cancause a fever.

Figure 1: About endotoxin and exotoxin (Midlands Technical College, 2012).

Characteristics and differences of exotoxins and endotoxin are listed below:

Characteristics Exotoxins EndotoxinsSource Living gram positive and gram

negative bacteriaLysed gram negative bacteria

Location Released from the cell Part of cellChemical composition Protein LipopolysaccarideHeat sensitivity Heat sensitive and inactivated at

60-80C. Toxicity destroyed over60C.

Heat stable to 250C. Toxicity isnot destroyed above 60C forhours.

Immune reactions Strong WeakConversion to toxoids Possible Not possibleFever Usually do not produce fever in

the host.

Usually produce fever in the host

by release of interleukin-1 andother mediators.

Specificity Specific to particular bacterialstrain

Non specific

Antigencity Highly antigenic. Stimulateformation of antitoxin. Antitoxinneutralizes the toxin.

Weakly antigenic. Antibodiesare protective.

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Examples Streptococcus Salmonella typhiTable: 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 responsiblefor its toxicity by binding to LBP and inducing the inflammatory cascade.

3. Lipooligosaccharide (LOS): It is a short chain endotoxin; found in N. meningitidis bacteriathat cause meningococcemia (Midlands Technical College, 2012).

The basic types of exotoxin based on structure and function:1. A-B toxins

2.

Membrane disrupting toxins3. Superantigens4. 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 ofSalmonella and E. coli . Lipopolysaccharides are complex amphiphilic molecules with a mw ofabout 10kDa, that vary widely in chemical composition both between and among bacterialspecies. 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

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Rd2 phenotype (not shown) would have only a single heptose unit. The Rc, Rd2, and Rd1mutants 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 INPHARMACEUTICAL 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 ananion exchanger. If the target substance is not also negatively charged, it will pass throughthe column before the endotoxin, and an effective separation can be achieved. Typicalexamples of endotoxin binding ligands include histamine, nitrogen-containing heterocycliccompounds, and polymyxin B.

2. Cation exchange chromatography : An alternative to anion exchange is cation exchangechromatography, in which positively charged solutes bind to the solid chromatographicmedia. In this method, the target binds to the column instead of the endotoxin. Theendotoxin 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

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the condensed vapor in an endotoxin free vessel. The large LPS molecules do not easilyvaporize, 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 sterilizeand /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/DESTRUCTIONOF 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. Thusunable to bind to endothelial cells, it is rendered inactive. However, acid-base hydrolysiscan 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 usedsafely and effectively. It is also widely used for depyrogenation of non-autoclavableequipment (e.g. plastics) and chromatography columns. In fact, when using an anionexchanger to remove pyrogens, it is necessary to clean the column with NaOH after each

batch.

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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 Availablefrom: 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 18thFebruary 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/ucm072918.htm [Accessed 18th February 2014]

http://classes.midlandstech.edu/carterp/Courses/bio225/chap15/lecture3.htmhttp://www.fda.gov/ICECI/Inspections/InspectionGuides/InspectionTechnicalGuides/ucm072918.htmhttp://www.fda.gov/ICECI/Inspections/InspectionGuides/InspectionTechnicalGuides/ucm072918.htmhttp://www.fda.gov/ICECI/Inspections/InspectionGuides/InspectionTechnicalGuides/ucm072918.htmhttp://www.fda.gov/ICECI/Inspections/InspectionGuides/InspectionTechnicalGuides/ucm072918.htmhttp://classes.midlandstech.edu/carterp/Courses/bio225/chap15/lecture3.htm