Endotoxin Testing is performed to ensure that injectable preparations and medical devices are free from pyrogens and safe for human use. Pyrogens constitute a heterogeneous group of fever causing substances which comprise both microbial and non-microbial substances. The most potent and most widely known are the endotoxins or lipopolysaccharides (LPS), which are cell wall components of gram-negative bacteria. Gram-positive bacteria are also sources of pyrogens, in particular lipoteichoic acid (LTA), as are particles from yeasts and viruses. Non-microbial pyrogens often emanate from production environments. Small particles of packaging materials are a typical example.

1. American Pharmaceutical Review – Endotoxin supplement
http://www.gibraltarlabsinc.com/endotoxin-testing.html
Selma Tarcan & Alvan Chao
Proprietary and Confidential © Gibraltar Laboratories, Inc.

2. American Pharmaceutical Review
Endotoxin Supplement 2015
Low Endotoxin Recovery, Sterile Products,
Endotoxin Detection, Rapid Methods,
Contamination Control

3. 3
Lecture AGENDA
Our course of discussion for today’s presentation
• General Information & History
• Definitions of endotoxin
• Detection Methods
• Rapid Methods
• Low Endotoxin Recovery
• Importance of Sterile Manufacturing
• Contamination Control

4. 4
Endotoxin
Purpose and History of Endotoxin Testing
• Where do Endotoxins come from ? Endotoxin contamination can occur through water, raw materials, media, equipment, containers used
in the manufacturing.
• Manufacturing process must be microbiologically controlled to reduce and remove endotoxins by monitoring raw materials and in process
intermediates at critical steps.
• Why do we perform endotoxin testing? Code of Federal Regulations 21CFR 211.167(a) requires that any drug product claimed to be
sterile and non-pyrogenic be tested prior to release!
• The necessity to ensure that parenteral products are free from heat stable endotoxins and other pyrogens was recognized during World
War II when the need for intravenous fluids was huge.
• At the time the safety was ensured by conducting Rabbit Pyrogen Test (Florence Seribert) RPT became part of USP in 1942.
• LAL (Limulus amebocyte lysate) is an aqueous extract of blood cells from the horseshoe crab, developed by Frederick Bang and Jack
Levin found that amebocytes of horseshoe crab will clot in the presence of endotoxins.
• LAL did not completely replaced RPT because of interferences with the LAL assay.

5. 5
Endotoxin
What are endotoxins?
• Components of the outer cell membranes of Gram-negative bacteria.
• Shed as part of the normal bacterial life cycle or by other processes that disrupt cells
• Contaminants in pharmaceutical raw materials, water systems, in process samples, and finished products.
• They are the major contributors to the pyrogenic response observed with contaminated pharmaceutical products
• Administration of parenteral products contaminated with pyrogens including LPS can lead to development of fever, induction of
inflammatory response, shock, organ failure and death in humans.
• Endotoxins contain lipopolysaccharide (LPS) molecules surrounded by surface proteins, lipoproteins and phospholipids.
• Lipopolysaccharides – Biologically active component of the endotoxin complex
• Located in the outer cell membrane of gram negative bacteria.
• Composed of three regions
• Lipid A: Active toxic portion. This region important for maintaining structural integrity.
• Core oligosaccharide: less heterogeneous and conserved. Inner core and outer core.
• O-antigen: consists of repeating units of glycosyl residue and structure varies among different bacterial strands.

6. 6
Endotoxin
Purpose, Test Method, and Definitions
• Naturally Occurring Endotoxin (NOE)
• NOEs are environmental, shedding from Gram negative bacteria.
• Control Standard Endotoxin (CSE)/Reference Standard Endotoxin (RSE)
• RSE  preparation supplied by the FDA
• RSE and CSE are used as calibration and test standards in BET.
• RSE and CSE are purified LPS and are distinguished from Natural Endotoxins.
• CSE (Control Standard Endotoxin) is a commercially prepared lyophilized endotoxin and its potency is
determined against RSE.
• Contains stabilizers like human serum albumin, Polyethylene Glycol, and starch
• CSE/RSE VS Endotoxin
• CSE and Endotoxin not considered to be the same. CSE – purified LPS
• LPS and endotoxin are physically, chemically and structurally different.

7. 7
Endotoxin
Test Methods - Bacterial Endotoxin Test (BET)
• Bacterial Endotoxin Test (BET) methods used:
• [USP <85> USP <151> USP <161>
• Gel-Clot method – qualitative method based on the formation of clotting of the test solution in presence of endotoxins
• Kinetic Chromogenic Method – quantitative method based on the color change of the reaction in the final test solution.
Measures the enzymatic reaction between bacterial endotoxin and the white blood cells of the horseshoe crab.
• Test compares sample or sample extract to standards prepared from Control Standard Endotoxin (CSE)
• Kinetic Chromogenic Test conducted by:
• Sample – test sample solution added to LAL reagent
• Positive Control - sample solution spiked with known amounts of CSE and added to LAL reagent.
• PPC Spike must recover 50-200% of CSE added
• <50% = Inhibition
• >200% = Enhancement

8. 8
Endotoxin
Test Methods - Bacterial Endotoxin Test (BET)
• As per USP <85> samples are prepared to “non interfering state” (dilution) where a nominal level of CSE activity can be quantitatively
recovered. (Inhibition/Enhancement or Validation)
• There is no requirement that CSE be recovered in undiluted products!
• Product interferences with LAL assay are known to occur as it as a highly sensitive test.
• Inhibition
• added known spike of CSE is “under estimated” activity is masked or diminished.
• Enhancement
• added known spike of CSE is “over estimated”
• Reagents used to overcome Inhibition/Enhancement: B-glucan blockers, cationic dispersing agents, divalent cations, heating.

9. 9
Endotoxin
Purpose, Test Method, and Definitions
• Low Endotoxin Recovery (LER)
• LER: “masking effect” caused by decline in the LAL reactivity to CSE after its addition to the test material.
• Is temperature dependent
• Primarily focus on Undiluted products
• Can be distinguished from interference commonly seen with the LAL assay that is normally overcome by dilution or other sample
preparation
• What causes LER?
• Precise mechanism of the LER matrix interference unknown but according to number of theories by industry researches there are
combination of factors that can cause LER.
• Polysorbate that is found in many protein formulations.
• Chelators
• Ionic nature of the protein
• Inhibition/Enhancement can be due to adsorption or aggregation, cation concentration, proteins.
FDA take on LER – Recommends hold time studies. FDA Q&A 2012 #3 “Firms should establish procedures for storing and
validating (which includes product mixing) samples of bacterial endotoxin analysis using laboratory data to demonstrate the
stability of assayable endotoxin content. Protocols should consider the source of endotoxin used in the study, bearing in mind that
endotoxins might react differently from native sources of endotoxins.
Why worry about LER? – contamination can occur during manufacturing process and finished product may contain endotoxins
(residual from the contamination) but if they cannot be detected by test methods, it can be a health and safety concern.

10. 10
Endotoxin
Test Methods
• Rabbit Pyrogen Test (RPT)
• RPT commonly used to detect endotoxin in products that is unable to be detected by LAL
method.
• in vivo [USP <151>] used to detect endotoxin in pharmaceutical products by monitoring the
increase in temperature or a fever following the intravenous injection of a test solution and is
designed for products that can be tolerated by the test rabbit in a dose not to exceed 10mL /kg
injected intravenously within a period of NMT 10 minutes.
• Limitations with RPT due to large number of use of animals for testing, continue to encourage
new methods.
• Rabbits may develop tolerance to endotoxin
• RPT may be waived if any other method demonstrated to be equivalent.

11. 11
Endotoxin
Alternative Methods – What other methods?
• Recombinant Factor C – delivers same reliability as LAL
• Activation of the first component in the LAL cascade
• No animal resources
• No false positive reactions from beta glucans
• Monocyte activation test (MAT).
• Alternative to rabbit pyrogen test
• Can test for
• Gram Negative
• Gram Positive
• Other biological pyrogens (eg yeast)
• Parasitic pyrogens
• Viral Pyrogens
• Uses cryopreserved human blood as source of monocytes (immune response cells)
• Monocytes recognize pyrogens and respond by releasing fever-inducing signal molecules
• Fever inducing molecules detected by ELISA.
• Eliminates interference from turbidity, color, or clotting
• Rapid Detection Methods
• Reduce cost, ease of use, time.
• Optical, Electrochemical, and mass based biosensors
• Still in Research and development

12. 12
Ph. Eur Sterile Products guidance
Future Update
• European guidelines under review – to revise annex 1 on manufacturing of sterile products
• Sterile manufacturing of pharmaceutical medicines plays an important role for microbiological contamination.
• Main risk concerns:
• Viable microorganisms
• Particulate matter
• Pyrogens (including bacterial endotoxin)
• Minimize risks
• Sterility achieved through
• Protective controls
• Good manufacturing practice
• Skill, training, and attitudes of personnel important.
• Quality assurance – to make sure carefully established and validated methods are strictly followed.

13. 13
Water for Pharmaceutical Use
US vs Ph. Eur – European guidelines under review
• USP vs Ph. Eur WFI water systems
• USP: WFI can be produced using either Distillation or Reverse Osmosis.
• Ph. Eur only permits Distillation to be used. May allow both after update.
• Distillation: process where component substances from a liquid mixture are separated
through the combination of selective evaporation and condensation
• Good for removing endotoxin
• Reverse Osmosis: uses a semi-permeable membrane to remove larger particles from
water through the application of an applied pressure.
• RO used to be less efficient
• Water for Pharmaceutical use typically Water for Injection

14. 14
Water
Water quality and usage

15. 15
Bioburden Contamination Control
Pharmaceuticals, medical devices and personal care product manufacturing
• To minimize product contamination and to monitor the environments within such
products are produced.
• All aspects of the operation should be designed to establish, implement and maintain
a quality system that ensures the delivery of pharmaceutical and health care products.
• Facilities
• Equipment
• Personnel
• Monitoring incoming raw materials
• Water systems
• Sanitizers

16. 16
Matrix
Overview of contamination control
Validation Control Monitoring
Facility Qualification of Clean
Room area and HVAC
system
Maintenance of facilities sanitization,
Revision of Barriers, Traffic Patterns, or
Air Balance
Environmental
Monitoring (EM)
HVAC Qualification of the Clean
Room area and HVAC
System
Certification and Preventative
Maintenance (PM) of System, Repair of
HEPA Filters
EM
Water Qualification of Water
System
Certification and PM Regular
Sanitization of System
Bioburden Monitoring
of Water System
Equipment Qualification of the
Equipment as Suitable for
its Intended Use
Certification and PM Regular
Sanitization
EM, Finished Product
Release Testing

17. 17
Matrix
Overview of contamination control
Validation Control Monitoring
Sanitization
Validation of Cleaning, Sanitization
and Sporicidal Treatments
Regular cleaning and
Sanitization of facilities and
equipment
EM
Personnel Proficiency criteria, participation in
media fills, trending data by
operator
Training Discipline
Personnel Monitoring
Trending Data by
Operator
Process
Process Validation
Acceptance Testing of Raw
Materials and Containers
In-Process Bioburden
Monitoring
Finished Product
Release Testing

18. 18
Conclusion
Overview of contamination control
• Some thoughts do not reflect those of FDA or other regulation
• LER is a concern of FDA and Biologics License Applications
• No cases associated with endotoxin contamination since 1987.
• Further research needed for LER, Rapid endotoxin, new technologies
• Guidance's under review
• Process controls for Endotoxin

19. 19
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